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The COVID-19 pandemic is a serious threat to global health and the global economy. The ongoing race to develop a safe and efficacious vaccine to prevent infection by SARS-CoV-2, the causative agent for COVID-19, highlights the importance of vaccination to combat infectious pathogens. The highly accessible cutaneous microenvironment is an ideal target for vaccination since the skin harbors a high density of antigen-presenting cells and immune accessory cells with broad innate immune functions. Microarray patches (MAPs) are an attractive intracutaneous biocargo delivery system that enables safe, reproducible, and controlled administration of vaccine components (antigens, with or without adjuvants) to defined skin microenvironments. This review describes the structure of the SARS-CoV-2 virus and relevant antigenic targets for vaccination, summarizes key concepts of skin immunobiology in the context of prophylactic immunization, and presents an overview of MAP-mediated cutaneous vaccine delivery. Concluding remarks on MAP-based skin immunization are provided to contribute to the rational development of safe and effective MAP-delivered vaccines against emerging infectious diseases, including COVID-19., Graphical abstract Unlabelled Image

Introduction Infectious pathogens are global disrupters. Progress in biomedical science and technology has expanded the public health arsenal against infectious diseases. Specifically, vaccination has reduced the burden of infectious pathogens. Engineering systemic immunity by harnessing the cutaneous immune network has been particularly attractive since the skin is an easily accessible immune-responsive organ. Recent advances in skin-targeted drug delivery strategies have enabled safe, patient-friendly, and controlled deployment of vaccines to cutaneous microenvironments for inducing long-lived pathogen-specific immunity to mitigate infectious diseases, including COVID-19. Areas covered This review briefly discusses the basics of cutaneous immunomodulation and provides a concise overview of emerging skin-targeted drug delivery systems that enable safe, minimally invasive, and effective intracutaneous administration of vaccines for engineering systemic immune responses to combat infectious diseases. Expert opinion In-situ engineering of the cutaneous microenvironment using emerging skin-targeted vaccine delivery systems offers remarkable potential to develop diverse immunization strategies against pathogens. Mechanistic studies with standard correlates of vaccine efficacy will be important to compare innovative intracutaneous drug delivery strategies to each other and to existing clinical approaches. Cost-benefit analyses will be necessary for developing effective commercialization strategies. Significant involvement of industry and/or government will be imperative for successfully bringing novel skin-targeted vaccine delivery methods to market for their widespread use.

The Parker Solar Probe and Solar Orbiter spacecraft make whole new spatial and time scales available in the inner Heliosphere. With these new data, we study directional discontinuities that are common structures in the solar wind in this region. Their radial distribution can provide insight into the physical processes of this virtually collisionless plasma. Applying a method (Erdős & Balogh, 2008) based on minimum variance analysis to select directional discontinuities in magnetic field data, we determine their number as a function of distance from the Sun. How the directional discontinuity occurrence rate depends on the solar wind speed is also part of our analysis.

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., Vaccine development against SARS‐CoV‐2 addresses the ongoing challenges posed by this pathogen and ensures preparedness for future outbreaks of coronaviruses. Subcutaneous or intranasal administration of Ad5.SARS‐CoV‐2 S1 results in multifaceted immune responses in mice, including GCs and long‐lived antibody forming cells, representing an attractive COVID‐19 vaccination strategy.

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 (SP) and hemokinin 1 (HK1), 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: To develop an Ag-specific immunosuppressive approach to treat CD by skin co-delivery 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-antagonists-MNA in CD. RESULTS: We demonstrate in mice that CD requires NK1R signaling by SP and HK1. Specific deletion of the NK1R in keratinocytes and dendritic cells, but not in mast cells prevented CD. Skin co-delivery of hapten- or Ag-NK1R-antagonist-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: Our findings demonstrate that immune-regulation by engineering localized skin neuroimmune networks can be used to treat cutaneous diseases that like CD, are caused by type-1 immunity.

Despite the role of donor-specific antibodies (DSAs) in recognizing major histocompatibility complex (MHC) antigens and mediating transplant rejection, how and where recipient B cells in lymphoid tissues encounter donor MHC antigens remains unclear. Contrary to the dogma, we demonstrated here that migration of donor leukocytes out of skin or heart allografts is not necessary for B or T cell allosensitization in mice. We found that mouse skin and cardiac allografts and human skin grafts release cell-free donor MHC antigens via extracellular vesicles (EVs) that are captured by subcapsular sinus (SCS) macrophages in lymph nodes or analog macrophages in the spleen. Donor EVs were transported across the SCS macrophages, and donor MHC molecules on the EVs were recognized by alloreactive B cells. This triggered B cell activation and DSA production, which were both prevented by SCS macrophage depletion. These results reveal an unexpected role for graft-derived EVs and open venues to interfere with EV biogenesis, trafficking, or function to restrain priming or reactivation of alloreactive B cells.

Dengue virus (DENV) and Zika virus (ZIKV) are closely related mosquito-borne flaviviruses that co-circulate in tropical regions and constitute major threats to global human health. Whether preexisting immunity to one virus affects disease caused by the other during primary or secondary infections is unknown but is critical in preparing for future outbreaks and predicting vaccine safety. Using a human skin explant model, we show that DENV-3 immune sera increased recruitment and infection of Langerhans cells, macrophages, and dermal dendritic cells following inoculation with DENV-2 or ZIKV. Similarly, ZIKV immune sera enhanced infection with DENV-2. Immune sera increased migration of infected Langerhans cells to the dermis and emigration of infected cells out of skin. Heterotypic immune sera increased viral RNA in the dermis almost 10-fold and reduced the amount of virus required to infect a majority of myeloid cells by 100- to 1000-fold. Enhancement was associated with cross-reactive IgG and induction of IL-10 expression and was mediated by both CD32 and CD64 Fcγ receptors. These findings reveal that preexisting heterotypic immunity greatly enhances DENV and ZIKV infection, replication, and spread in human skin. This relevant tissue model will be valuable in assessing the efficacy and risk of dengue and Zika vaccines in humans.

The skin is an attractive tissue target for vaccination, as it is readily accessible and contains a dense population of antigen-presenting and immune-accessory cells. Microneedle arrays (MNAs) are emerging as an effective tool for in situ engineering of the cutaneous microenvironment to enable diverse immunization strategies. Here, we present novel dissolving undercut MNAs and demonstrate their application for effective multicomponent cutaneous vaccination. The MNAs are composed of micron-scale needles featuring pyramidal heads supported by undercut stem regions with filleted bases to ensure successful skin penetration and retention during application. Prior efforts to fabricate dissolving undercut microstructures were limited and required complex and lengthy processing and assembly steps. In the current study, we strategically combine three-dimensional (3D) laser lithography, an emerging micro-additive manufacturing method with unique geometric capabilities and nanoscale resolution, and micromolding with favorable materials. This approach enables reproducible production of dissolving MNAs with undercut microneedles that can be tip-loaded with multiple biocargos, such as antigen (ovalbumin) and adjuvant (Poly(I:C)). The resulting MNAs fulfill the geometric (i.e., sharp tips and smooth edges) and mechanical-strength requirements for failure-free penetration of human and murine skin to simultaneously deliver multicomponent (antigen plus adjuvant) vaccines to the same cutaneous microenvironment. Cutaneous vaccination of mice using these MNAs induces more potent antigen-specific cellular and humoral immune responses than those elicited by traditional intramuscular injection. Together, the unique geometric features of these undercut MNAs and the associated manufacturing strategy, which is compatible with diverse drugs and biologics, could enable a broad range of non-cutaneous and cutaneous drug delivery applications, including multicomponent vaccination.

Contact dermatitis (CD) is a chronic inflammatory disease caused by type-1 immunity. Skin exposure to haptens stimulates the secretion of Substance-P (SP) and initiates the neurogenic inflammation that intensifies CD. Neurokinin-1 receptor (NK1R)-signaling by SP or hemokinin-1 (HK1) amplifies immune responses. Nonetheless, the role and therapeutic implications of the NK1R-SP-HK1 axis in CD remain unclear. We show that SP, HK-1 and the NK1R are required for CD. Specific deletion of the NK1R in keratinocytes decreased the rapid release of IL-1β and IL-6 at the site of contact sensitization which impaired the innate and adaptive immunity of CD whereas deletion of the receptor in dendritic cells (DC) prevented only the adaptive immune response of the disease. Therefore, we hypothesized that blockade of NK1R during sensitization would be a feasible immunosuppressive intervention to treat CD. We developed a system of microneedle arrays (MNA) that co-deliver hapten and NK1R antagonists into mouse skin. This immunosuppressive approach resulted in decreased skin migration and lymph node homing of stimulatory dermal DC transporting the hapten from the sensitization site. Conversely, the immunosuppressive MNA did not affect the migration and lymph node homing of epidermal Langerhans cells (LC), and depletion of LC resulted in loss of the NK1R antagonist beneficial effects. In addition, immunosuppressive MNA caused deletion of hapten-specific T cells and increased T-regulatory cells, which prevented CD-onset and -relapses in a hapten-specific manner. Our findings indicate that immune-regulation by engineering localized skin neuroimmune-networks can be used to treat cutaneous diseases that, like CD are caused by type-1 immunity.

Considerable progress has been made toward development of COVID-19 vaccines in the past year However, there is still a need for painless, self-administered, shelf-stable, and efficacious SARS-CoV-2 vaccines to enable sustainable immunization programs against COVID-19 To address this need, we hypothesized that harnessing the immune-responsive cutaneous microenvironment using microarray patches (MAPs) to deliver integrated SARS-CoV-2 vaccine components would bring together biologic advantages of targeting the endogenous immune circuitry of the skin with a thermostable and user-friendly cutaneous vaccine delivery platform We show that immunologically rich cutaneous microenvironments in both murine and human skin can be efficiently targeted using our 3D printing-enabled dissolving MAPs to deliver a recombinant SARS-CoV-2 protein antigen, with or without an innate immune agonist Immunization of mice with vaccine-loaded MAPs generates robust antibody and cellular immune responses, and multicomponent (antigen plus adjuvant) MAP vaccination improves the induced antigen-specific immune responses, such as virus-specific Th1 and IgG2c responses, which are vital for control of SARS-CoV-2 viral infection Notably, multicomponent MAP vaccination results in increased immune responses compared to immunization via traditional intramuscular injection, and MAP immunization obviates adverse effects of intramuscular delivery of adjuvants, suggesting improved safety and efficacy compared to conventional vaccination routes These results are supported by our translational studies utilizing freshly-excised human skin, suggesting that multicomponent MAPs induce greater expression of co-stimulatory molecules by human skin-migratory DCs, which may contribute to enhanced immune responses Ultimately, the simplicity, thermostability, immunogenicity, and versatility of MAPs may enable novel vaccination strategies and increase the effectiveness of global immunization campaigns against SARS-CoV-2 and other existing or novel pathogens

Autoinflammatory skin diseases are characterized by a disequilibrium of cytokines in the local skin microenvironment, suggesting that local delivery of therapeutics, including anticytokine antibodies, may provide benefit without the unwanted off-target effects of systemically delivered therapies. Rapid diffusion of therapeutics away from the target site has been a challenge to the development of local therapies. Conjugation of high molecular weight hydrophilic polymers to cytokine neutralizing mAbs has been shown to be an effective strategy for local control of inflammation in healing burn wounds. However, the burn application is unique because the skin barrier is already breached. For the treatment of autoinflammatory skin diseases, the major challenge for local delivery lies in penetrating the stratum corneum. Here, we investigate a new therapeutic approach combining the use of tip-loaded dissolvable microneedle arrays (TL-dMNAs) for local application of polymer-conjugated antibody inhibitors of tumor-necrosis-factor-alpha (TNF-α). Specifically, intradermal delivery and pharmacokinetics of (anti-TNF-α-Ab)-(high molecular weight hyaluronic acid [HA]) conjugates from tip-loaded, obelisk-shaped dissolvable microneedle arrays were investigated in living human skin. The results indicate (1) TL-dMNAs can be successfully fabricated to integrate (anti-TNF-α-Ab)-HA at the tip portion of the microneedles while preserving the biological activity necessary for antibody ligand binding; (2) (anti-TNF-α-Ab)-HA can be effectively delivered into human skin using obelisk-shaped TL-dMNAs; and (3) polymer conjugation effectively inhibits antibody diffusion from the delivery site. Taken together, these results support the evaluation of microneedle array-based delivery of varying polymer-antibody conjugates for the treatment of inflammatory skin diseases.

Substance P (SP) is a proinflammatory neuropeptide that following Ag entrance in peripheral tissues signals via the neurokinin 1 receptor (NK1R) to initiate innate and support adaptive cellular immune responses. These mechanisms underlie chronic skin inflammatory disorders like contact dermatitis (CD). Here we propose to develop an immunosuppressive method to prevent and treat CD by blocking the effects of SP during skin sensitization with haptens. We utilized microneedle arrays to efficiently deliver the hapten 2,4-dinitrocholorobencene (DNCB) and NK1R antagonists simultaneously to the skin of C57/BL6 mice. This approach, restrained neuroinflammation, increased T regulatory cells and decreased the viability of Th1 and Tc1 biased cells in the draining lymph nodes. Together these effects inhibited local and systemic CD and prevented its relapses. Using the Cre-Lox P system, we demonstrate that specific deletion of the NK1R in keratinocytes but not in leukocytes inhibited the sensitization phase of CD by blocking the release of IL-1β and IL-6. Whereas deletion of the receptor in keratinocytes or in dendritic cells was necessary to abrogate the adaptive cellular immunity. Our data demonstrate the possibility of preventing the development of cellular immunity by engineering the skin microenvironment to restrain the effects of neuroinflammatory peptides accounting for the onset of chronic skin inflammatory diseases.

Background Coronaviruses pose a serious threat to global health as evidenced by Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and COVID-19. SARS Coronavirus (SARS-CoV), MERS Coronavirus (MERS-CoV), and the novel coronavirus, previously dubbed 2019-nCoV, and now officially named SARS-CoV-2, are the causative agents of the SARS, MERS, and COVID-19 disease outbreaks, respectively. Safe vaccines that rapidly induce potent and long-lasting virus-specific immune responses against these infectious agents are urgently needed. The coronavirus spike (S) protein, a characteristic structural component of the viral envelope, is considered a key target for vaccines for the prevention of coronavirus infection. Methods We first generated codon optimized MERS-S1 subunit vaccines fused with a foldon trimerization domain to mimic the native viral structure. In variant constructs, we engineered immune stimulants (RS09 or flagellin, as TLR4 or TLR5 agonists, respectively) into this trimeric design. We comprehensively tested the pre-clinical immunogenicity of MERS-CoV vaccines in mice when delivered subcutaneously by traditional needle injection, or intracutaneously by dissolving microneedle arrays (MNAs) by evaluating virus specific IgG antibodies in the serum of vaccinated mice by ELISA and using virus neutralization assays. Driven by the urgent need for COVID-19 vaccines, we utilized this strategy to rapidly develop MNA SARS-CoV-2 subunit vaccines and tested their pre-clinical immunogenicity in vivo by exploiting our substantial experience with MNA MERS-CoV vaccines. Findings Here we describe the development of MNA delivered MERS-CoV vaccines and their pre-clinical immunogenicity. Specifically, MNA delivered MERS-S1 subunit vaccines elicited strong and long-lasting antigen-specific antibody responses. Building on our ongoing efforts to develop MERS-CoV vaccines, promising immunogenicity of MNA-delivered MERS-CoV vaccines, and our experience with MNA fabrication and delivery, including clinical trials, we rapidly designed and produced clinically-translatable MNA SARS-CoV-2 subunit vaccines within 4 weeks of the identification of the SARS-CoV-2 S1 sequence. Most importantly, these MNA delivered SARS-CoV-2 S1 subunit vaccines elicited potent antigen-specific antibody responses that were evident beginning 2 weeks after immunization. Interpretation MNA delivery of coronaviruses-S1 subunit vaccines is a promising immunization strategy against coronavirus infection. Progressive scientific and technological efforts enable quicker responses to emerging pandemics. Our ongoing efforts to develop MNA-MERS-S1 subunit vaccines enabled us to rapidly design and produce MNA SARS-CoV-2 subunit vaccines capable of inducing potent virus-specific antibody responses. Collectively, our results support the clinical development of MNA delivered recombinant protein subunit vaccines against SARS, MERS, COVID-19, and other emerging infectious diseases.

Allergic contact dermatitis (ACD) is a common T-cell mediated inflammatory skin condition, characterized by an intensely pruritic rash at the site of contact with allergens like poison ivy or nickel. Current clinical treatments use topical corticosteroids, which broadly and transiently suppress inflammation and symptoms of ACD, but fail to address the underlying immune dysfunction. Here, we present an alternative therapeutic approach that teaches the immune system to tolerate contact allergens by expanding populations of naturally suppressive allergen-specific regulatory T cells (Tregs). Specifically, biodegradable poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PEG-PLGA) microparticles were engineered to release TGF-β1, Rapamycin, and IL-2, to locally sustain a microenvironment that promotes Treg differentiation. By expanding allergen-specific Tregs and reducing pro-inflammatory effector T cells, these microparticles inhibited destructive hypersensitivity responses to subsequent allergen exposure in an allergen-specific manner, effectively preventing or reversing ACD in previously sensitized mice. Ultimately, this approach to in vivo Treg induction could also enable novel therapies for transplant rejection and autoimmune diseases.

The skin is the site of dengue virus (DENV) transmission following the bite of an infected mosquito, but the contribution of individual cell types within skin to infection is unknown. We studied the dynamics of DENV infection in human skin explants using quantitative in situ imaging. DENV replicated primarily in the epidermis and induced a transient IFN-α response. DENV infected a wide range of cells, including Langerhans cells, macrophages, dermal dendritic cells, mast cells, fibroblasts, and lymphatic endothelium, but keratinocytes were the earliest targets of infection and made up 60% of infected cells over time. Virus inoculation led to recruitment and infection of Langerhans cells, macrophages, and dermal dendritic cells, and these cells emigrated from skin in increased numbers as a result of infection. DENV induced expression of proinflammatory cytokines and chemokines by infected keratinocytes. Blocking keratinocyte-derived IL-1β alone reduced infection of Langerhans cells, macrophages, and dermal dendritic cells by 75−90% and reduced the overall number of infected cells in dermis by 65%. These data show that the innate response of infected keratinocytes attracts virus-permissive myeloid cells that inadvertently spread DENV infection. Our findings highlight a role for keratinocytes and their interplay with myeloid cells in dengue.

The immune response against transplanted allografts is one of the most potent reactions mounted by the immune system. The acute rejection response has been attributed to donor dendritic cells (DCs), which migrate to recipient lymphoid tissues and directly activate alloreactive T cells against donor MHC molecules. Here, using a murine heart transplant model, we determined that only a small number of donor DCs reach lymphoid tissues and investigated how this limited population of donor DCs efficiently initiates the alloreactive T cell response that causes acute rejection. In our mouse model, efficient passage of donor MHC molecules to recipient conventional DCs (cDCs) was dependent on the transfer of extracellular vesicles (EVs) from donor DCs that migrated from the graft to lymphoid tissues. These EVs shared characteristics with exosomes and were internalized or remained attached to the recipient cDCs. Recipient cDCs that acquired exosomes became activated and triggered full activation of alloreactive T cells. Depletion of recipient cDCs after cardiac transplantation drastically decreased presentation of donor MHC molecules to directly alloreactive T cells and delayed graft rejection in mice. These findings support a key role for transfer of donor EVs in the generation of allograft-targeting immune responses and suggest that interrupting this process has potential to dampen the immune response to allografts.

Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs down-regulate T-cell responses to induce/maintain immunologic tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles (< 100 nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle miRNAs and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, a hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, because they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and posttranscriptional regulation between DCs.

Hot flow anomalies (HFAs) are studied using observations of the RAPID suprathermal charged particle detector, the FGM magnetometer, and the CIS plasma detector aboard the four Cluster spacecraft. Previously, we studied several specific features of tangential discontinuities on the basis of Cluster measurements in February-April 2003. In this paper, we confirm the following results: the angle between the Sun direction and the tangentional discontinuity (TD) normal is larger than 45{\deg} during HFAs, the magnetic field directional change is large. We then present evidence for a new necessary condition for the formation of HFAs, that is, the solar wind speed is significantly ( about 200 km/s or dMf=2.3) higher than the long-term average. The existence of this condition is also confirmed by simultaneous ACE MAG and SWEPAM solar wind observations at the L1 point 1.4 million km upstream of the Earth. The results are compared with recent hybrid simulations., Comment: 11 pages, 6 figures, published in 2008

Tumor necrosis factor-alpha (TNF-α) specific antibodies (anti-TNF-α Ab) have been shown to be potent TNF inhibitors and effective therapeutics for a range of inflammatory diseases. Typically, these drugs are administered systemically, but systemic dosing sufficient to achieve locally effective concentrations in peripheral tissues has been associated with systemic immunosuppression and related adverse events. Here, we evaluated the use of tip-loaded dissolvable microneedle arrays (MNAs) for localized intradermal delivery of anti-TNF-α Ab. MNAs with obelisk shape microneedles that incorporate the antibody cargo in the needle tips were created from carboxymethylcellulose (CMC) using a micromilling/spin-casting fabrication method. We found that anti-TNF-α Ab integrated into MNAs using this room temperature fabrication process maintained conformationally dependent TNF-α binding activity. Further, these MNAs efficiently delivered anti-TNF-α antibodies to the dermis of human skin with clinically applicable release profiles. To evaluate MNA delivered anti-TNF-α Ab function, we applied anti-TNF-α Ab containing MNAs to established psoriasiform lesions on the skin of mice. MNA anti-TNF-α Ab treatment reduced key biomarkers of psoriasiform inflammation including epidermal thickness and IL-1β expression. Taken together, these results demonstrate efficient and biologically effective MNA delivery of anti-TNF-α Ab to the intradermal microenvironment of the skin in mice and humans, and support the development of MNA mediated antibody delivery for clinical applications. Statement of Significance Tumor necrosis factor-alpha (TNF-α) specific antibodies (anti-TNF-α Ab) have been shown to be potent TNF inhibitors and effective therapeutics for a range of inflammatory diseases. Typically, these drugs are administered systemically, but systemic dosing sufficient to achieve locally effective concentrations in peripheral tissues has been associated with systemic immunosuppression and related adverse events. Here we demonstrate efficient and biologically effective MNA delivery of anti-TNF-α Ab to the intradermal microenvironment of the skin in mice and humans. These results support the development of MNA mediated antibody delivery of therapeutic antibodies for clinical applications.

The distributed genome hypothesis (DGH) states that each strain within a bacterial species receives a unique distribution of genes from a population-based supragenome that is many times larger than the genome of any given strain. The observations that natural infecting populations are often polyclonal and that most chronic bacterial pathogens have highly developed mechanisms for horizontal gene transfer suggested the DGH and provided the means and the mechanisms to explain how chronic infections persist in the face of a mammalian host's adaptive defense mechanisms. Having previously established the validity of the DGH for obligate pathogens, we wished to evaluate its applicability to an opportunistic bacterial pathogen. This was accomplished by construction and analysis of a highly redundant pooled genomic library containing approximately 216,000 functional clones that was constructed from 12 low-passage clinical isolates of Pseudomonas aeruginosa , 6 otorrheic isolates and 6 from other body sites. Sequence analysis of 3,214 randomly picked clones (mean insert size, ∼1.4 kb) from this library demonstrated that 348 (10.8%) of the clones were unique with respect to all genomic sequences of the P. aeruginosa prototype strain, PAO1. Hypothetical translations of the open reading frames within these unique sequences demonstrated protein homologies to a number of bacterial virulence factors and other proteins not previously identified in P. aeruginosa . PCR and reverse transcription-PCR-based assays were performed to analyze the distribution and expression patterns of a 70-open reading frame subset of these sequences among 11 of the clinical strains. These sequences were unevenly distributed among the clinical isolates, with nearly half (34/70) of the novel sequences being present in only one or two of the individual strains. Expression profiling revealed that a vast majority of these sequences are expressed, strongly suggesting they encode functional proteins.

Skin dendritic cells (DC) are professional APC critical for initiation and control of adaptive immunity. In the present work we have analyzed the CD4+ T cell stimulatory function of different subsets of DC that migrate spontaneously from human skin explants, including CD1a+CD14− Langerhans’ cells (LC), CD1a−CD14− dermal DC (DDC), and CD1a−CD14+ LC precursors. Skin migratory DC consisted of APC at different stages of maturation-activation that produced IL-10, TGF-β1, IL-23p19, and IL-12p40, but did not release IL-12p70 even after exposure to DC1-driving stimuli. LC and DDC migrated as mature/activated APC able to stimulate allogeneic naive CD4+ T cells and to induce memory Th1 cells in the absence of IL-12p70. The potent CD4+ T cell stimulatory function of LC and DDC correlated with their high levels of expression of MHC class II, adhesion, and costimulatory molecules. The Th1-biasing function of LC and DDC depended on their ability to produce IL-23. By contrast, CD1a−CD14+ LC precursors migrated as immature-semimature APC and were weak stimulators of allogeneic naive CD4+ T cells. However, and opposite of a potential tolerogenic role of immature DC, the T cell allostimulatory and Th1-biasing function of CD14+ LC precursors increased significantly by augmenting their cell number, prolonging the time of interaction with responding T cells, or addition of recombinant human IL-23 in MLC. The data presented in this study provide insight into the function of the complex network of skin-resident DC that migrate out of the epidermis and dermis after cutaneous immunizations, pathogen infections, or allograft transplantation.

Dendritic cells are professional antigen-presenting cells capable of inducing and regulating innate and antigen-specific immune responses. Therapeutic cancer vaccines using ex vivo engineered or in vivo targeted dendritic cells are being evaluated in clinical trials. T-helper type-1 (Th1)–skewed immune responses are characterized by the preferential induction of antigen-specific IFN-γ–secreting CD4+ T cells and correlate with effector mechanisms important for tumor and viral immunity. Methods to “polarize” human monocyte-derived dendritic cells for the preferential induction of Th1-skewed immune responses have been developed, and polarized dendritic cells (DC1s) are being evaluated in preclinical and clinical studies. Here, we show that stimulation of bone marrow–derived murine dendritic cell populations with poly(I:C) and CpGs results in phenotypic maturation of dendritic cells and synergistic induction of durable, high-level IL-12p70 secretion characteristic of human type-1 polarized dendritic cells. Functionally, these dendritic cells induce antigen-specific Th1-type CD4+ T-cell activation in vitro and in vivo. Dendritic cell maturation and polarization are not inhibited by the presence of live B16 melanoma tumor cells, and tumor-loaded DC1s induce delayed-type hypersensitivity responses in vivo. DC1s loaded with B16 melanoma cells and injected into tumor-bearing mice induce Th1-skewed tumor-specific CD4+ T cells and a significant reduction in tumor growth. Tumor infiltrates in DC1-immunized animals are characterized by the presence of CD4+ T cells and activated macrophages. These results show a murine model of DC1 function and suggest an important role for CD4+ T cells and macrophages in DC1-induced antitumor immune responses. They have implications for the future development of DC1-based immunotherapies and strategies for clinical immune monitoring of their effectiveness.

Haemophilus influenzae is one of the most important respiratory pathogens of man. It has been etiologically associated with otitis media, otorrhea, and chronic obstructive pulmonary disease. Identification of new genomic elements will provide novel targets to fight chronic infections caused by this organism. Objective: The new paradigm that chronic infections are caused by bacterial biofilms prompted us to study the relationship between bacterial pathogenicity, biofilm formation and bacterial communal cooperation. To do this, it is essential to determine the virulence gene sets that are involved in the above processes and whether they are present in every bacterial cell or distributed in a “communal gene-pool”, the distributed genome hypothesis (DGH). We designed, constructed and characterized a highly redundant genomic DNA library comprised of the genomes of ten low passage clinical isolates of H. influenzae carrying large numbers of genes that are not present in the laboratory strains of H. influenzae . Methods: Genomic DNA fragments of the ten clinical strains were hydro-dynamically sheared to produce a mean fragment size of 1.5–2.5 kb. The ten sheared DNAs were than pooled and used in the construction of a genomic library with 76 800 clones. Results: Our restriction endonuclease and sequence analyses of 800 clones demonstrate that 75% of the clones carry an insert larger than 0.5 kb. The library has an ∼1.5 kb average insert size, and therefore, better than 4.5× redundancy for each of the genomes of the ten clinical isolates. Our sequencing effort (∼1 million nucleotides to date) reveals that a high percentage of genes (75 clones, 11% of the 686 sequenced clones) present in this library are not represented in the genome of the reference strain H. influenzae Rd. Conclusions: The library, based on the above results, has a better than 4.5× coverage for each of the ten constituent genomes. On the basis of our preliminary sequencing data (∼1 million nucleotides) the library lacks of highly repeated sequences, therefore, the expected genome coverage (4.5×) is not degraded. Using the prevalence of non-Rd like sequences (11%) detected during characterization of the genomic library, we estimated that the library contains DNA sequences equivalent to ∼2 million bp, which are not represented in the reference genome of the H. influenzae Rd strain and that is greater in size than the genome of this reference strain, providing ample targets for innovative drug design.

We have previously demonstrated that microneedle array (MNA) delivery of anti-tumor agents improves survival in mouse models of melanoma and non-melanoma skin cancers and induces cell death in freshly excised human cutaneous squamous cell carcinomas (cSCCs). As tumors employ strong immunosuppressive and tumor-promoting mechanisms that prevent their elimination by immune surveillance mechanisms and support their proliferation, we sought to determine if application of MNAs containing anti-tumor agents could abrogate their protumorigenic phenotype and promote an anti-tumor phenotype in a human model of cSCC, the second most common human malignancy worldwide. To accomplish this, we compared Nanostring gene expression data from human cSCCs treated with MNAs containing doxorubicin (MNA-Dox) to matched, untreated cSCC controls and unmatched normal skin. As compared to matched, untreated cSCC controls, MNA-Dox-treated cSCCs demonstrated significantly decreased expression of genes associated with a pro-tumorigenic environment, including IDO, ARG2, S100A7 and VEGF-A. Further, MNA-Dox-treated cSCCs demonstrated significantly increased expression of genes associated with an anti-tumor phenotype, including CXCL14, HMGB1, P53, and Caspase-8. Comparison of MNA-Dox-treated cSCCs and unmatched, untreated normal skin suggested a reversion of a pro-tumorigenic phenotype to a normal skin phenotype, with similarly low expression of pro-tumorigenic genes seen in both groups. Together, these results suggest the potential of MNA-administered doxorubicin to effectively transform a pro-tumor microenvironment to one favoring the induction of tumor immunity in a human cSCC.

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.

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.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.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.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.

We studied the effect of ionizing radiation on the activation of the AP-1 transcription factors and the regulation of basic fibroblast growth factor (bFGF) gene expression in drug-sensitive human breast carcinoma (MCF-7) cells and its drug-resistant variant (MCF-7/ADR) cells. Northern blot and gel mobility shift assays showed that 135 cGy of ionizing radiation induced c-jun and c-fos gene expression, AP-1 binding activity, as well as bFGF gene expression in MCF-7/ADR cells. In MCF-7 cells, however, we observed little/no induction of bFGF gene expression and AP-1 binding activity after the stress. Nevertheless, MCF-7 cells transfected with plasmids containing c-jun gene contain high levels of bFGF protein. H-7 (60 micrograms/ml), a potent protein kinase C (PKC) inhibitor, inhibited the stress-induced AP-1 binding activity and bFGF gene expression in MCF-7/ADR cells. Corroborating this observation, overexpression of PKC alpha induced bFGF gene expression in MCF-7 cells. Taken together, these results suggest that stress-induced bFGF gene expression is mediated through the activation of PKC and AP-1 transcription factors. Differences in the levels of PKC activity and AP-1 binding factors may be responsible for differential expression of bFGF among breast cancer cell lines. Although there are large differences in response to ionizing radiation between MCF-7 and MCF-7/ADR cell lines, we observed no significant differences in radiocytotoxicity between them.

We studied the effect of isoquinolinesulfonamide derivatives (H-7, H-8, and HA 1004) on the expression of two heat shock genes ( αB - crystallin and hsp70 ) in NIH 3T3 and Swiss 3T3 cells after heat shock at 45° for 10 min. Western blots and northern blots showed that H-7 effectively suppressed the accumulation of HSP70 and αB-crystallin mRNA as well as the synthesis of their proteins. The degree of suppression was dependent upon the concentration of the drug. Moreover, the expression of the hsp genes was differentially suppressed by H-7. The expression of the αB - crystallin gene was more effectively inhibited than that of the hsp70 gene by H-7. Nuclear run-on assay demonstrates that this difference was due to the differential effect of H-7 on the elongation of transcription of different hsp genes.

We have developed a vaccine design involving the conjugation of polyguanine (Poly(dG)) to protein or peptide antigens for the specific targeting of scavenger receptors in dendritic cells with intrinsic adjuvant capabilities. We have shown that Poly(dG) conjugation to ovalbumin generates stable particle conjugates (Poly(dG)-OVA) that induce greater T cell (helper, cytotoxic, and memory) and antibody responses. In this study, we show that Poly(dG)-OVA treatment of DCs in vitro induces greater antigen internalization and expression of activation markers CD86, MHC II, and CCR7. In vivo, intradermal injection of Poly(dG)-OVA resulted in the formation of an antigen depot in the skin lasting for several days and an increase in recruited immune infiltrates. Increased association of Poly(dG)-OVA is observed with MHC II- and CD11c-expressing cells in the skin compared to OVA. This is accompanied by the expression of pro-inflammatory CCL2 and IL-6 in the skin. Poly(dG)-OVA was also able to internalize more in skin migrating DCs and was transported more efficiently to draining lymph nodes. Poly(dG)-OVA immunization of B16-OVA-bearing mice contributed to delayed tumor progression and improved survival. We also developed Poly(dG)-Mgp10025–33 and Poly(dG)-TRP2180–188 to study melanoma-specific responses. Immunization with the these conjugates led to significant peptide-specific in vivo cytotoxic T cell responses compared to unconjugated peptides. Mice with B16 tumors immunized with combined Poly(dG)-Mgp100 Poly(dG)-TRP2 had delayed tumor progression and improved survival. Our results show that Poly(dG) conjugation of antigens is an effective strategy for the development of antitumor vaccines with improved immune responses.

We have previously demonstrated that dissolvable microneedle arrays can co-deliver cargos directly to the skin while avoiding systemic distribution. As squamous cell carcinoma (SCC) has a lifetime incidence of 7–11% in the United States and is the second most common non-melanoma skin cancer in persons of white European descent, we sought to determine if MNA delivery of anti-tumor agents induce cell death and improve survival in a mouse model of SCC and in freshly excised human SCCs. To accomplish this, we evaluated the effectiveness of doxorubicin, a chemotherapeutic demonstrated to induce immunogenic cell death, as an MNA delivered therapeutic. Effectiveness was evaluated in mice inoculated with SCC cells and later treated with Blank MNAs (MNA-Blank) or MNAs integrating doxorubicin (MNA-Dox), and in human SCCs treated with MNA-Dox. In mice, MNA-Dox treatment resulted in increased levels of pro-inflammatory cytokine gene expression, increased levels of cell death as determined by TUNEL assays, attenuated tumor growth, and substantially improved survival over MNA-Blank treatment at 20 days post-inoculation (100% survival in MNA-Dox group versus 0% survival in MNA-Blank group). Survival advantage persisted with 40% of MNA-Dox-treated mice alive at 40 days post-inoculation. Similarly, freshly excised human SCCs treated with MNA-Dox demonstrated increased levels of cell death as determined by TUNEL assays. Taken together, these results demonstrate the potential of MNA administration of doxorubicin to effectively induce tumor cell death in a murine model of squamous cell carcinoma and in human SCCs supporting further clinical development.

This paper describes an ultra-compliant parylene-platinum neural probe embedded in a biodissolvable delivery vehicle. High probe compliance is achieved using thin wires (width of 10.0 μm and thickness of 2.7 μm) and by meandering the probe. The insertion of the ultra-compliant probe is achieved by encasing it in a dissolvable delivery vehicle made from molded carboxy-methylcellulose. In vivo implantations of delivery vehicles with 1.5 mm long shanks, widths of 100 μm and 300 μm and a targeted thickness of 135 μm have been done through the dura in the cortex of Sprague-Dawley rats at a speed of 80 mm-s−1. The delivery vehicle becomes a gel over a period of less than three minutes, after which the handling portions of the delivery vehicle are removed leaving the shanks embedded in the brain.

The effect of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent protein kinase C (PKC) inhibitor, on the development of thermotolerance and expression of heat shock genes (HSP70 and HSP28) was investigated in human colon carcinoma HT-29 cells. After acute heating at 45 degrees for 15 min, cells became resistant to a challenge heat shock. The development of thermotolerance was suppressed by adding H-7 after heat shock. Northern blots show that the levels of HSP70 and HSP28 mRNA increased rapidly and reached maximal values within 6 hr. H-7 suppressed the accumulation of HSP70 and HSP28 mRNA as well as their protein synthesis, and the level of suppression was concentration dependent. However, little effect was observed if the drug was added 1 hr before and during heat shock. These results suggest that PKC is involved in the regulation of heat shock gene expression after acute heat shock.

1. 1|Treatment with cycloheximide (CHM; 10 μg/ml) 2 h before and during heating produced a 13-fold increase in survival from 1 × 10 −2 to 1.3 × 10 −1 after 10h at 43°C in human colon carcinoma HT-29 cells. Little protection was observed if the drug was added only during heating. 2. 2|Pretreatment with 10 μg/m CHM which inhibited protein synthesis by 95%, decreased the level of nascent polypeptides and subsequently reduced the accumulation of polypeptides in the nucleus during heat shock. 3. 3|Adding CHM (10 μg/ml) 2h before and during heat facilitated the dissociation of heat shock transcription factor-heat shock element (HSF-HSE) complex and consequently terminated transcription activity earlier. 4. 4|These findings related to the literature suggest that the free pool of HSP70 is increased by inhibiting protein synthesis. An increase in the level of free HSP70 may more effectively protect or repair thermolabile targets and consequently affect the regulation of heat shock response.

1. 1.|While human breast carcinoma (MCF-7) cells were resistant to heat shock, human prostatic carcinoma (DUT-145 and PC-3) cells were relatively sensitive to mild hyperthermia. For example, survival of both prostatic carcinoma cells or that of breast carcinoma cells was 10 or 93%, respectively, after heating at 41°C for 24h. 2. 2.|We investigated whether or not heat shock gene expression might be responsible for these differences in thermal sensitivity. 3. 3.|It was observed that transcriptional activation of heat shock genes was induced by heating at 41°C in all three cell lines. Studies from the gel mobility shift assay demonstrated the formation of heat shock factor and heat shock element (HSF-HSE) complex during heating at 41°C for 2 h in both MCF-7 and DUT-145 cell lines. 4. 4.|In addition, Northern blots and polyacrylamide gel electrophoresis demonstrated expression of heat shock genes, along with the synthesis of their proteins, particularly 68 kDa heat shock protein in all three cell lines. 5. 5.|These results demonstrate that differences in thermal sensitivity to mild hyperthermia are not due to differential heat shock gene expression.

The possible involvement of PKC in the regulation of heat shock genes expression was investigated with three isoquinolinesulfonamide derivatives (H-7, H-8, and HA1004) in DUT-145, MCF-7, and MCF-7/ADR cells. The drug was added 1 hr before and during heating at 41°C. Northern blots show that the levels of HSP70 and HSP28 mRNA increased rapidly and reached maximal values within 4-8 hr and 8-12 hr, respectively. H-7 and H-8 which are potent PKC inhibitors selectively suppressed the accumulation of HSP70 mRNA as well as the synthesis of HSP70. In contrast, HA1004 which is a potent PKA inhibitor but a weak PKC inhibitor did not affect HSP70 gene expression. These results suggest that PKC rather than PKA plays an important role in the regulation of heat shock gene expression.

The prevailing idea regarding the mechanism(s) by which therapeutic immunosuppressive dendritic cells (DCs) restrain alloimmunity is based on the concept that they interact directly with antidonor T cells, inducing anergy, deletion, and/or regulation. However, this idea has not been tested in vivo. Using prototypic in vitro–generated maturation-resistant (MR) DCs, we demonstrate that once MR-DCs carrying donor antigen (Ag) are administered intravenously, they decrease the direct and indirect pathway T-cell responses and prolong heart allograft survival but fail to directly regulate T cells in vivo. Rather, injected MR-DCs are short-lived and reprocessed by recipient DCs for presentation to indirect pathway CD4+ T cells, resulting in abortive activation and deletion without detrimental effect on the number of indirect CD4+ FoxP3+ T cells, thus increasing the regulatory to effector T cell relative percentage. The effect on the antidonor response was independent of the method used to generate therapeutic DCs or their viability; and in accordance with the idea that recipient Ag-presenting cells mediate the effects of therapeutic DCs in transplantation, prolongation of allograft survival was achieved using donor apoptotic MR-DCs or those lacking surface major histocompatibility complex molecules. We therefore conclude that therapeutic DCs function as Ag-transporting cells rather than Ag-presenting cells to prolong allograft survival.

Hot flow anomalies (HFAs) are studied using observations of the FGM magnetometer and the CIS plasma detector aboard the four Cluster spacecraft. Previously we studied several specific features of tangential discontinuities on the basis of Cluster measurements in February-April 2003 and discovered a new condition for forming HFAs that is the solar wind speed is higher than the average. However during the whole spring season of 2003, the solar wind speed was higher than average. In this study we analyse HFAs detected in 2007, the year of solar cycle minimum. Our earlier result was confirmed: the higher solar wind speed is a real condition for HFA formation; furthermore this constraint is independent of Schwartz et al. s condition for HFA formation.