Solid nanoemulsion as antigen and immunopotentiator carrier for transcutaneous immunization.

Imiquimod, a toll-like receptor 7 (TLR7) agonist, is an active pharmaceutical ingredient (API) established for the topical treatment of several dermal cancerous and precancerous skin lesions. Within this work, the immunostimulatory effect of imiquimod is further exploited in a transcutaneous immunization (TCI) approach based on a solid nanoemulsion (SN) formulation. SN contains a combination of imiquimod with the model peptide antigen SIINFEKL as a novel approach to omit needle and syringe and optimize dermal antigen administration. Excipients including sucrose fatty acid esters and the pharmaceutically acceptable oils MCT (middle chain triglycerides), avocado oil, jojoba wax and squalene are high pressure homogenized together with the antigen SIINFEKL. Freeze drying was performed to eliminate water and to achieve spreadable properties of the formulation for dermal administration. The influence of the different oil components was assessed regarding in vitro drug permeation in a Franz diffusion cell model using a murine skin setup. In vivo performance in terms of cytotoxic T-cell response was assessed in a C57BL/6 mouse model. Whereas Aldara® cream contains imiquimod in a dissolved state, the SN formulations carry the active in a suspended state. This resulted in a reduction of imiquimod permeation across murine skin from the SN when compared to Aldara® cream. In spite of this permeation rate reduction, each SN induced an in vivo immune response by specific T-cell lysis. A stabilized solid nanosuspension containing squalene/tocopherol exhibited a significantly higher performance (p⩽0.05) in comparison with Aldara® cream. MCT based SN exerted an in vivo effect comparable to Aldara®. In conclusion, anhydrous highly dispersed vehicles containing imiquimod in a submicron particle size distribution can represent promising formulations for TCI. The choice of the oil component has a strong influence on SN performance, independent of in vitro drug permeation.

Evaluation and Validation of the Detection of soluble Triggering Receptor Expressed on Myeloid Cells 1 by Enzyme-linked immunosorbent Assay.

Triggering receptor expressed on myeloid cells (TREM)-1 plays an important role in innate immune responses and is upregulated under infectious as well as non-infectious conditions. In addition, a soluble TREM-1 variant (sTREM-1) is detectable in sera or bronchoalveolar-lavage fluids from patients. Currently, various studies are difficult to compare, since the methods of detection by enzyme-linked immunosorbent assays (ELISA) vary among different research groups. In this study, we compared three different s-TREM-1 specific ELISAs and identified individual assay characteristics finding notable differences in sTREM-1 concentrations in part depending on the employed buffers. Investigating potential confounding factors for sTREM-1 detection, serum heat-inactivation (HI) showed improved recovery compared to non-HI (NHI) serum, reproducible by addition of complement and re-heat-inactivation. Hence we identified complement as a heat-sensitive confounder in some sTREM-1 ELISAs. We conclude that it is difficult to directly compare data of several studies, in particular if different ELISAs are engaged. Immunoassays for research use only are in general hampered by lack of standardization. Further standardization is needed until sTREM-1 ELISA is capable for better reproducibility of studies and clinical application.

Change of walking distance in intermittent claudication: impact on inflammation, oxidative stress and mononuclear cells: a pilot study.

BACKGROUND: Atherosclerosis is a chronic inflammatory process involving the immune system and formation of reactive oxygen species (ROS). We investigated changes of mononuclear blood cells and ROS production in relation to the walking distance of patients with intermittent claudication during home-based exercise training. METHODS: Forty patients with intermittent claudication were asked to perform a home-based exercise training for a mean time of 12 months. ROS formation was measured using the luminol analogue L-012. Peripheral blood leucocytes [monocytes, polymorphonuclear neutrophils (PMN) and dendritic cells (DC)] were analysed by flow cytometry and analysed for the expression of major inflammatory surface molecules. RESULTS: At follow-up, patients showed an increased walking distance and reduced ROS production upon stimulation with a phorbol ester derivative (PDBu) (p < 0.01). Monocytes changed their inflammatory phenotype towards an increased anti-inflammatory CD14(++)CD16(-) subpopulation (p < 0.0001). Adhesion molecules CD11b, CD11c and TREM-1 on monocytes and PMN decreased (all p < 0.01). On DC expression of HLA-DR, CD86 or CD40 decreased at follow-up. Inflammatory markers like fibrinogen, C-reactive protein or soluble TREM-1 (sTREM-1) decreased over the observation period. Finally, we found a close relation of sTREM-1 with the walking distance, fibrinogen and ROS production. CONCLUSIONS: We observed an amelioration of the proinflammatory phenotype on monocytes, DC and PMN, as well as a reduced ROS production in PAD patients under home-based exercise, paralleled by an increased walking distance. Our data suggest that a reduced inflammatory state might be achieved by regular walking exercise, possibly in a dimension proportionately to changes in walking distance.

Cofactor-independent antiphospholipid antibodies activate the NLRP3-inflammasome via endosomal NADPH-oxidase: implications for the antiphospholipid syndrome.

The antiphospholipid syndrome (APS) is an autoimmune disease characterised by thromboembolic events and/or pregnancy morbidity in the presence of antiphospholipid antibodies (aPL). Here we show that three cofactor independent human monoclonal aPL can induce transcription of NLRP3 and caspase-1 resulting in inflammasome activation specific for NLRP3. This depends fully on activation of endosomal NADPH-oxidase-2 (NOX2) by aPL. Activation of NOX2 and subsequent inflammasome activation by aPL are independent from TLR2 or TLR4. While endosomal superoxide production induces caspase-1 and NLRP3 transcription, it does not affect prae-IL-1ß transcription. Therefore, release of IL-1ß occurs only after activation of additional pathways like TLR7/8 or TLR2. All effects exerted by the monoclonal aPL can be reproduced with IgG fractions of APS patients proving that the monoclonal aPL are representative for the APS. IgG fractions of healthy controls or patients suffering from systemic lupus erythematosus have no effect. In a mouse model of the APS we can show inflammasome activation in vivo. Furthermore, mononuclear cells isolated from patients with the APS show an increased expression of caspase-1 and NLRP3 which is accompanied by a three-fold increased serum concentration of IL-1ß suggesting chronic inflammasome activation in APS patients. In summary, we provide further evidence that endosomal NOX2 can be activated by cofactor independent aPL. This leads to induction of the NLRP3 inflammasome. Our data indicate that cofactor independent aPL might contribute significantly to the pathogenesis of the APS.

Perceptions of oral health in Appalachian Kentucky: implications for message design.

We conducted three studies to investigate Appalachian college students' perceptions and behavior concerning oral health for the purpose of identifying salient factors to consider in designing persuasive messages to promote oral health. In Study 1, we conducted seven focus groups with 67 college students at a state university in Appalachian Kentucky. Using thematic analysis, we found that students based their oral health behavior on the perception that people living in Appalachia have poor oral health and that students denied, confirmed, reframed, or fulfilled this "misconception." In Study 2, quantitative results from a representative survey of students (N = 587) at the same university indicated that the barriers to enacting good oral health behavior were primarily logistical in nature, whereas the facilitators of good oral health behavior were largely social. In Study 3, results from dental screenings (N = 364) of students at the university demonstrated that about one in five students presented with active, visible decay. We discuss how these results inform our understanding of oral health behavior in Appalachia and the implications of these results for designing messages to promote oral health in the region.

Dental Care in the Frail Older Adult: Special Considerations and Recommendations.

Frail older adults disproportionately suffer from untreated dental problems. Age-related biological changes to hard and soft dental tissues, existing medical conditions, polypharmacy, diet and uncontrolled plaque exacerbate the problem. All of these factors increase the complexity of treatment and will differ greatly from standard treatment of younger adults. This article discusses the key considerations and suggestions for risk assessment, disease management, treatment planning and palliative care to maintain the patient's comfort and quality of life.

Host-derived CD8⁺ dendritic cells protect against acute graft-versus-host disease after experimental allogeneic bone marrow transplantation.

Graft-versus-host disease (GVHD) is a frequent life-threatening complication after allogeneic hematopoietic stem cell transplantation (HSCT) and induced by donor-derived T cells that become activated by host antigen-presenting cells. To address the relevance of host dendritic cell (DC) populations in this disease, we used mouse strains deficient in CD11c(+) or CD8α(+) DC populations in a model of acute GVHD where bone marrow and T cells from BALB/c donors were transplanted into C57BL/6 hosts. Surprisingly, a strong increase in GVHD-related mortality was observed in the absence of CD11c(+) cells. Likewise, Batf3-deficient (Batf3(-/-)) mice that lack CD8α(+) DCs also displayed a strongly increased GVHD-related mortality. In the absence of CD8α(+) DCs, we detected an increased activation of the remaining DC populations after HSCT, leading to an enhanced priming of allogeneic T cells. Importantly, this was associated with reduced numbers of regulatory T cells and transforming growth factor-ß levels, indicating an aggravated failure of peripheral tolerance mechanisms after HSCT in the absence of CD8α(+) DCs. In summary, our results indicate a critical role of CD8α(+) DCs as important inducers of regulatory T cell-mediated tolerance to control DC activation and T cell priming in the initiation phase of GVHD.

Efficacy of imiquimod-based transcutaneous immunization using a nano-dispersed emulsion gel formulation.

BACKGROUND: Transcutaneous immunization (TCI) approaches utilize skin associated lymphatic tissues to elicit specific immune responses. In this context, the imidazoquinoline derivative imiquimod formulated in Aldara applied onto intact skin together with a cytotoxic T lymphocyte (CTL) epitope induces potent CTL responses. However, the feasibility and efficacy of the commercial imiquimod formulation Aldara is limited by its physicochemical properties as well as its immunogenicity. METHODOLOGY/PRINCIPAL FINDINGS: To overcome these obstacles, we developed an imiquimod-containing emulsion gel (IMI-Gel) and characterized it in comparison to Aldara for rheological properties and in vitro mouse skin permeation in a Franz diffusion cell system. Imiquimod was readily released from Aldara, while IMI-Gel showed markedly decreased drug release. Nevertheless, comparing vaccination potency of Aldara or IMI-Gel-based TCI in C57BL/6 mice against the model cytotoxic T-lymphocyte epitope SIINFEKL, we found that IMI-Gel was equally effective in terms of the frequency of peptide-specific T-cells and in vivo cytolytic activity. Importantly, transcutaneous delivery of IMI-Gel for vaccination was clearly superior to the subcutaneous or oral route of administration. Finally, IMI-Gel based TCI was at least equally effective compared to Aldara-based TCI in rejection of established SIINFEKL-expressing E.G7 tumors in a therapeutic setup indicated by enhanced tumor rejection and survival. CONCLUSION/SIGNIFICANCE: In summary, we developed a novel imiquimod formulation with feasible pharmaceutical properties and immunological efficacy that fosters the rational design of a next generation transcutaneous vaccination platform suitable for the treatment of cancer or persistent virus infections.

Donor and host B cell-derived IL-10 contributes to suppression of graft-versus-host disease.

Graft-versus-host disease (GvHD) is a frequent life-threatening complication following allogeneic HSC transplantation (HSCT). IL-10 is a regulatory cytokine with important roles during GvHD, yet its relevant sources, and mode of action, remain incompletely defined in this disease. Using IL-10-deficient donor or host mice (BALB/c or C57BL/6, respectively) in a MHC-mismatched model for acute GvHD, we found a strongly aggravated course of the disease with increased mortality when either donor or host cells could not produce this cytokine. A lack of IL-10 resulted in increased allogeneic T-cell responses and enhanced activation of host DCs in spleen and MLNs. Remarkably, IL-10 was prominently produced by host- and donor-derived CD5(int) CD1d(int) TIM-1(int) B cells in this disease, and consistent with this, allogeneic HSCT resulted in exacerbated GvHD when mice lacking IL-10 expression in B cells were used as donor or host, compared with controls. Taken together, this study demonstrates that host and donor B cell-derived IL-10 provides a unique mechanism of suppression of acute GvHD, and suggests that DCs are the targets of this B cell-mediated suppressive effect. These findings open novel therapeutic possibilities based on the use of B cells to increase the feasibility of allogeneic HSCT.

Strategies for communicating with older dental patients.

BACKGROUND: Communication between dentists and patients 65 years or older is a critical aspect of providing optimum care, particularly given the increasing number of older adults and the communication barriers they often encounter. METHODS: The authors conducted a targeted literature review of the broad health communication literature and published health literacy guidelines to examine the barriers to effective communication that are specific to the older adult population, as well as strategies for overcoming these barriers. RESULTS: Findings from health communication and health literacy research provide insight into techniques to improve communication with older patients, such as preparing an agenda for the appointment, exhibiting warm nonverbal behavior, listening attentively, asking open-ended questions, using simple language, presenting key points one at a time and providing patients with written instructions. CONCLUSIONS: Physical, psychological and literacy issues pertaining to both patients and providers present barriers to effective communication. Practitioners can surmount these barriers by enacting communication strategies tailored to older adults. Practical Implications. Dentists can overcome barriers to communication and improve the quality of patient care by considering the communication barriers specific to older adults and enacting strategies to overcome these barriers.

Distinct signaling cascades of TREM-1, TLR and NLR in neutrophils and monocytic cells.

Triggering receptor expressed on myeloid cells 1 (TREM-1) is an important mediator of innate inflammatory responses in microbial infections and sepsis. TREM-1 ligation on neutrophils (PMN) or monocytes results in the production of proinflammatory cytokines. Engagement of TREM-1 induces the activation of MAP kinases as well as rapid Ca(2+) mobilization. However, a detailed understanding of TREM-1 signaling pathways is currently lacking. We evaluated the TREM-1 signaling hierarchy in monocytic cells and found that the acute myeloid leukemia cell line MUTZ-3 expresses TREM-1 in a natural and functional manner. We compared essential signaling molecules of the TREM-1, TLR and NLR cascade in MUTZ-3 cells as well as primary monocytes or PMN by Western blot analysis. These studies confirmed the essential role of phosphatidyl inositide 3-kinase (PI3K) and p38MAPK in the TREM-1 as well as the TLR or NLR cascade of monocytic cells. Importantly, PI3K and p38MAPK signals in monocytic cells both control Ca(2+) mobilization and are directly connected in the TREM-1 signaling hierarchy, which contrasts previous results obtained in PMN. Taken together, our results indicate cell type-specific differences in the TREM-1 signaling cascade and contribute to an enhanced understanding of the regulation of innate inflammatory responses.

Oxidative burst and neutrophil elastase contribute to clearance of Aspergillus fumigatus pneumonia in mice.

Polymorphonuclear neutrophils (PMN) are important for the control of invasive aspergillosis (IA), a major threat to immunocompromised individuals. For clearance of Aspergillus fumigatus infections, PMN employ their potent oxidative and non-oxidative mechanisms. To clarify the relative contribution of these mechanisms, we analyzed p47(phox-/-), gp91(phox-/-) and elastase (ELA) deficient mice (ELANE) after intratracheal infection with A. fumigatus. Infected p47(phox-/-) and gp91(phox-/-) mice died within 4 days and had a significant higher fungal burden in the lungs compared to wild-type controls. Interestingly, the survival of ELANE mice after infection was unimpaired suggesting that ELA is not essential here. Nevertheless, A. fumigatus clearance was delayed in ELANE mice indicating a partial contribution of ELA to fungal immunity. Comparing p47(phox-/-), gp91(phox-/-) or ELANE mice for PMN activation and recruitment to the lungs, we were unable to detect significant differences in vitro or in vivo among mutant or wild-type strains suggesting intact PMN functionality of basic effector mechanisms. Fungal killing in vitro by ELA deficient PMN was comparably reduced as in p47(phox-/-) and gp91(phox-/-) deficient PMN corroborating the importance of oxidative and non-oxidative PMN mechanisms for the control of fungal outgrowth. Taken together, this suggests that intact oxidative as well as non-oxidative PMN effector functions are highly relevant for the control of A. fumigatus infections in vitro and in vivo. While ELA contributes to clearance of A. fumigatus, the oxidative functions are essential for survival.

Dental pain in the ED: costs that hurt patients and EDs.

BACKGROUND AND OBJECTIVES: The objective of this retrospective study was to determine if the collection rates for dental related visits to the emergency department (ED) are less than collection rates for ED visits for other problems. METHODS: Data were analyzed from one Kentucky hospital's electronic health record system from April 2010 to April 2012. Collection rates for patients who received care in the ED for uncomplicated dental problems were compared to collection rates for all patients who received care in the ED for any reason. RESULTS: Each month during the study period, an average of 77 patients presented to the ED for dental problems. Compensation rates for physician fees were 9.8% for dental related care and 39% for all patients who received care for any reason. Compensation rates for hospital fees were 16% for dental related care and 20.1% for all patients who received care for any reason. Uninsured patients accounted for 68.8% of physician fees and 62.4% of hospital fees for dental related care. CONCLUSIONS: Using the ED as a dental safety net is costly to the patient because the underlying problem is typically not resolved and costly to the hospital because of very low collection rates. In addition, other patients who present to the ED for non-dental, high acuity problems may have delayed care or no care because of the number of patients using the ED for dental pain.

Mechanisms of cyclic nucleotide phosphodiesterases in modulating T cell responses in murine graft-versus-host disease.

Graft-versus-host disease (GvHD) is a key contributor to the morbidity and mortality after allogeneic hematopoetic stem cell transplantation (HSCT). Regulatory Foxp3(+) CD4(+) T cells (Treg) suppress conventional T cell activation and can control GvHD. In our previous work, we demonstrate that a basic mechanism of Treg mediated suppression occurs by the transfer of cyclic adenosine monophosphate (cAMP) to responder cells. Whether this mechanism is relevant for Treg mediated suppression of GvHD is currently unknown. To address this question, bone marrow and T cells from C57BL/6 mice were transferred into lethally irradiated BALB/c recipients, and the course of GvHD and survival were monitored. Transplanted recipients developed severe GvHD that was strongly ameliorated by the transfer of donor Treg cells. Towards the underlying mechanisms, in vitro studies revealed that Treg communicated with DCs via gap junctions, resulting in functional inactivation of DC by a metabolic pathway involving cAMP that is modulated by the phosphodiesterase (PDE) 4 inhibitor rolipram. PDE2 or PDE3 inhibitors as well as rolipram suppressed allogeneic T cell activation, indirectly by enhancing Treg mediated suppression of DC activation and directly by inhibiting responder T cell proliferation. In line with this, we observed a cooperative suppression of GvHD upon Treg transfer and additional rolipram treatment. In conclusion, we propose that an important pathway of Treg mediated control of GvHD is based on a cAMP dependent mechanism. These data provide the basis for future concepts to manipulate allogeneic T cell responses to prevent GvHD.

Steady-state neutrophil homeostasis is dependent on TLR4/TRIF signaling.

UNLABELLED: Polymorphonuclear neutrophil granulocytes (neutrophils) are tightly controlled by an incompletely understood homeostatic feedback loop adjusting the marrow's supply to peripheral needs. Although it has long been known that marrow cellularity is inversely correlated with G-CSF levels, the mechanism linking peripheral clearance to production remains unknown. Herein, the feedback response to antibody induced neutropenia is characterized to consist of G-CSF­dependent shifts of marrow hematopoietic progenitor populations including expansion of the lin-/Sca-1/c-kit (LSK) and granulocyte macrophage progenitor (GMP) compartments at the expense of thrombopoietic and red cell precursors. Evidence is provided that positive feedback regulation is independent from commensal germs as well as T, B, and NK cells. However, in vivo feedback is impaired in TLR4-/- and TRIF-/-, but not MyD88-/- animals. In conclusion, steady-state neutrophil homeostasis is G-CSF­dependent and regulated through pattern-recognition receptors,thereby directly linking TLR-triggering to granulopoiesis. KEY POINTS: Steady-state and emergency granulopoiesis are both dependent on TLR signaling.

A role for Toll-like receptor mediated signals in neutrophils in the pathogenesis of the anti-phospholipid syndrome.

The anti-phospholipid syndrome (APS) is characterized by recurrent thrombosis and occurrence of anti-phospholipid antibodies (aPL). aPL are necessary, but not sufficient for the clinical manifestations of APS. Growing evidence suggests a role of innate immune cells, in particular polymorphonuclear neutrophils (PMN) and Toll-like receptors (TLR) to be additionally involved. aPL activate endothelial cells and monocytes through a TLR4-dependent signalling pathway. Whether this is also relevant for PMN in a similar way is currently not known. To address this issue, we used purified PMN from healthy donors and stimulated them in the presence or absence of human monoclonal aPL and the TLR4 agonist LPS monitoring neutrophil effector functions, namely the oxidative burst, phagocytosis, L-Selectin shedding and IL-8 production. aPL alone were only able to induce minor activation of PMN effector functions at high concentrations. However, in the additional presence of LPS the activation threshold was markedly lower indicating a synergistic activation pathway of aPL and TLR in PMN. In summary, our results indicate that PMN effector functions are directly activated by aPL and boosted by the additional presence of microbial products. This highlights a role for PMN as important innate immune effector cells that contribute to the pathophysiology of APS.

Serum antibodies to periodontal pathogens are a risk factor for Alzheimer's disease.

BACKGROUND: Chronic inflammation in periodontal disease has been suggested as a potential risk factor in Alzheimer's disease (AD). The purpose of this study was to examine serum antibody levels to bacteria of periodontal disease in participants who eventually converted to AD compared with the antibody levels in control subjects. METHODS: Serum samples from 158 participants in the Biologically Resilient Adults in Neurological Studies research program at the University of Kentucky were analyzed for immunoglobulin G antibody levels to seven oral bacteria associated with periodontitis, including Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Campylobacter rectus, Treponema denticola, Fusobacterium nucleatum, Tannerella forsythia, and Prevotella intermedia. All 158 participants were cognitively intact at baseline venous blood draw. In all, 81 of the participants developed either mild cognitive impairment (MCI) or AD or both, and 77 controls remained cognitively intact in the years of follow-up. Antibody levels were compared between controls and subjects with AD at baseline draw and after conversion and controls and subjects with MCI at baseline draw and after conversion using the Wilcoxon rank-sum test. AD and MCI participants were not directly compared. Linear regression models were used to adjust for potential confounding. RESULTS: Antibody levels to F nucleatum and P intermedia were significantly increased (α = 0.05) at baseline serum draw in the patients with AD compared with controls. These results remained significant when controlling for baseline age, Mini-Mental State Examination score, and apolipoprotein epsilon 4 status. CONCLUSIONS: This study provides initial data that demonstrate elevated antibodies to periodontal disease bacteria in subjects years before cognitive impairment and suggests that periodontal disease could potentially contribute to the risk of AD onset/progression. Additional cohort studies profiling oral clinical presentation with systemic response and AD and prospective studies to evaluate any cause-and-effect association are warranted.

Comparative transcutaneous immunization with imiquimod-containing ointments and potential of in vitro methods to predict effects.

This work evaluates the transcutaneous in vitro and in vivo immunization efficacy of five commercially available 5% imiquimod containing formulations. The parameters included microscopic analysis, rheological properties, drug permeation across synthetic membranes of molecular weight cutoff 10 kDa and ablated murine skin with both 0.1 m HCl and a phthalate buffer pH 3.6 Ph.Eur./methanol 3/7 (v/v) as receiver solutions in a Franz-diffusion cell model. For in vivo formulation characterization, the cytotoxic T-cell activity and interferon-γ production in C57BL/6 mice was determined ex vivo 24 h after transcutaneous administration. OVA(257-264) (SIINFEKL) from chicken albumin served as a target antigen. Microscopic images demonstrated differences with respect to the presence or absence of crystalline API. Rheological properties point to a roughly ten-fold difference between the products at low shear rates. With regard to drug permeation across synthetic membranes, only 'Nan Bo' demonstrated equality compared with Aldara™, resulting in f1 and f2 values of 5.25 and 59.58, respectively. The drug permeation rates were maximum from Aldara™ across ablated murine skin. Furthermore, differentiation between the formulations containing crystalline and dissolved states of active imiquimod was possible using this model. The in vivo results yielded significant immunomodulating activities (p < 0.05) of multisource formulations compared with the untreated control group, however, the significance of differences between the formulations was dependent on the parameter of interest. A correlation plot of skin permeation versus in vivo immunostimulating activity yielded a slope significantly different from zero only in the case of the murine skin setup (r between 0.421 and 0.669). Yet this correlation is deemed not satisfactory for formulation optimization.