Biofilm-coated microbeads and the mouse ear skin: An innovative model for analysing anti-biofilm immune response in vivo.

Owing to its ability to form biofilms, Staphylococcus aureus is responsible for an increasing number of infections on implantable medical devices. The aim of this study was to develop a mouse model using microbeads coated with S. aureus biofilm to simulate such infections and to analyse the dynamics of anti-biofilm inflammatory responses by intravital imaging. Scanning electron microscopy and flow cytometry were used in vitro to study the ability of an mCherry fluorescent strain of S. aureus to coat silica microbeads. Biofilm-coated microbeads were then inoculated intradermally into the ear tissue of LysM-EGFP transgenic mice (EGFP fluorescent immune cells). General and specific real-time inflammatory responses were studied in ear tissue by confocal microscopy at early (4-6h) and late time points (after 24h) after injection. The displacement properties of immune cells were analysed. The responses were compared with those obtained in control mice injected with only microbeads. In vitro, our protocol was capable of generating reproducible inocula of biofilm-coated microbeads verified by labelling matrix components, observing biofilm ultrastructure and confirmed in vivo and in situ with a matrix specific fluorescent probe. In vivo, a major inflammatory response was observed in the mouse ear pinna at both time points. Real-time observations of cell recruitment at injection sites showed that immune cells had difficulty in accessing biofilm bacteria and highlighted areas of direct interaction. The average speed of cells was lower in infected mice compared to control mice and in tissue areas where direct contact between immune cells and bacteria was observed, the average cell velocity and linearity were decreased in comparison to cells in areas where no bacteria were visible. This model provides an innovative way to analyse specific immune responses against biofilm infections on medical devices. It paves the way for live evaluation of the effectiveness of immunomodulatory therapies combined with antibiotics.

Peri-arterial Autonomic Innervation of the Human Ear.

Auricular vasomotor responses are considered to be signs of clinical conditions including migraine. The mechanisms of auricular vasomotor control are still debatable. This study aimed at investigating perivascular co-transmitters of vasomotor control in the auricle. Another aim was to provide three-dimensional arterial maps of the auricle, as a proxy of periarterial autonomic innervation. Twelve paired human auricles were used to visualize the arteries following Spalteholz clearing and µ-CT-based reconstruction. Perivascular innervation staining was conducted using anti-tyrosine hydroxylase (TH), anti-neuropeptide Y (NPY), anti-vasoactive intestinal peptide (VIP) and anti-choline acetyl transferase (ChAT). The combined Spalteholz technique and µ-CT revealed a highly consistent arrangement of the auricular vasculature. The superficial temporal (STA) and posterior auricular artery (PAA) supply the helical rim arcade and arcade, with the STA mainly forming the superior and the PAA forming the middle and inferior auricular artery. Co-existence of sympathetic NPY+ and TH+ terminals mediating vasoconstriction, and VIP+ and ACh+ indicating cholinergic vasodilatation, was found in the perivascular zone. The presence of both sympathetic vasoconstriction and cholinergic co-innervation for active vasodilatation was shown in the perivascular auricular zone. Assuming that the highly-consistent vasculature gives way to these terminals, this periarterial innervation may be found spread out across the helix.

Non-Invasive Multiphoton Imaging of Islets Transplanted Into the Pinna of the NOD Mouse Ear Reveals the Immediate Effect of Anti-CD3 Treatment in Autoimmune Diabetes.

We present a novel and readily accessible method facilitating cellular time-resolved imaging of transplanted pancreatic islets. Grafting of islets to the mouse ear pinna allows non-invasive, in vivo longitudinal imaging of events in the islets and enables improved acquisition of experimental data and use of fewer experimental animals than is possible using invasive techniques, as the same mouse can be assessed for the presence of islet infiltrating cells before and after immune intervention. We have applied this method to investigating therapeutic protection of beta cells through the well-established use of anti-CD3 injection, and have acquired unprecedented data on the nature and rapidity of the effect on the islet infiltrating T cells. We demonstrate that infusion of anti-CD3 antibody leads to immediate effects on islet infiltrating T cells in islet grafts in the pinna of the ear, and causes them to increase their speed and displacement within 20 min of infusion. This technique overcomes several technical challenges associated with intravital imaging of pancreatic immune responses and facilitates routine study of beta islet cell development, differentiation, and function in health and disease.

Role of the K(Ca)3.1 K+ channel in auricular lymph node CD4+ T-lymphocyte function of the delayed-type hypersensitivity model.

BACKGROUND AND PURPOSE: The intermediate-conductance Ca(2+)-activated K(+) channel (K(Ca)3.1) modulates the Ca(2+) response through the control of the membrane potential in the immune system. We investigated the role of K(Ca)3.1 on the pathogenesis of delayed-type hypersensitivity (DTH) in auricular lymph node (ALN) CD4(+) T-lymphocytes of oxazolone (Ox)-induced DTH model mice. EXPERIMENTAL APPROACH: The expression patterns of K(Ca)3.1 and its possible transcriptional regulators were compared among ALN T-lymphocytes of three groups [non-sensitized (Ox-/-), Ox-sensitized, but non-challenged (Ox+/-) and Ox-sensitized and -challenged (Ox+/+)] using real-time polymerase chain reaction, Western blotting and flow cytometry. KCa 3.1 activity was measured by whole-cell patch clamp and the voltage-sensitive dye imaging. The effects of K(Ca)3.1 blockade were examined by the administration of selective K(Ca)3.1 blockers. KEY RESULTS: Significant up-regulation of K(Ca)3.1a was observed in CD4(+) T-lymphocytes of Ox+/- and Ox+/+, without any evident changes in the expression of the dominant-negative form, K(Ca)3.1b. Negatively correlated with this, the repressor element-1 silencing transcription factor (REST) was significantly down-regulated. Pharmacological blockade of K(Ca)3.1 resulted in an accumulation of the CD4(+) T-lymphocytes of Ox+/+ at the G0/G1 phase of the cell cycle, and also significantly recovered not only the pathogenesis of DTH, but also the changes in the K(Ca)3.1 expression and activity in the CD4(+) T-lymphocytes of Ox+/- and Ox+/+. CONCLUSIONS AND IMPLICATIONS: The up-regulation of K(Ca)3.1a in conjunction with the down-regulation of REST may be involved in CD4(+) T-lymphocyte proliferation in the ALNs of DTH model mice; and K(Ca)3.1 may be an important target for therapeutic intervention in allergy diseases such as DTH.

Label-free 3D imaging of microstructure, blood, and lymphatic vessels within tissue beds in vivo.

This Letter reports the use of an ultrahigh resolution optical microangiography (OMAG) system for simultaneous 3D imaging of microstructure and lymphatic and blood vessels without the use of an exogenous contrast agent. An automatic algorithm is developed to segment the lymphatic vessels from the microstructural images based on the fact that the lymph fluid is optically transparent. An OMAG system is developed that utilizes a broadband supercontinuum light source, providing an axial resolution of 2.3 µm and lateral resolution of 5.8 µm, capable of resolving the capillary vasculature and lymphatic vessels innervating microcirculatory tissue beds. Experimental demonstration is performed by showing detailed 3D lymphatic and blood vessel maps, coupled with morphology, within mouse ears in vivo.

Infection of mice with Francisella as an immunological model.

This unit describes the utility of various mouse models of infection for studying pathogenesis and adaptive immune responses to the facultative intracellular bacteria pathogen Francisella tularensis. By judicious use of different combinations of mouse and bacterial strains, as well as different routes of infection, murine tularemia models may be used to explore a complete picture of F. tularensis infection and immunity. Moreover, studies using Francisella, particularly the Live Vaccine Strain (LVS), serve as a convenient and tractable model system that appears to be representative of mammalian host responses to intracellular pathogens in general.

Transcutaneous immunization as preventative and therapeutic regimens to protect against experimental otitis media due to nontypeable Haemophilus influenzae.

We have developed three nontypeable Haemophilus influenzae (NTHI) adhesin-derived immunogens that are significantly efficacious against experimental otitis media (OM) due to NTHI when delivered parenterally. We now expanded our preventative immunization strategies to include transcutaneous immunization (TCI) as a less invasive, but potentially equally efficacious, regimen to prevent OM due to NTHI. Additionally, we examined the potential of TCI as a therapeutic immunization regimen to resolve ongoing experimental OM. Preventative immunization with NTHI outer membrane protein (OMP) P5- and type IV pilus-targeted immunogens, delivered with the adjuvant LT(R192G-L211A), induced significantly earlier clearance of NTHI from the nasopharynges and middle ears of challenged chinchillas compared with receipt of immunogen or adjuvant alone. Moreover, therapeutic immunization resulted in significant resolution of established NTHI biofilms from the middle ear space of animals compared with controls. These data advocate TCI with the adhesin-directed immunogens as an efficacious regimen for prevention and resolution of experimental NTHI-induced OM.

Immunological response to tissue-engineered cartilage derived from auricular chondrocytes and a PLLA scaffold in transgenic mice.

The immune response against biomaterials in tissue-engineered constructs could potentially worsen the outcome of tissue regeneration, but immunological reactions between host and donor in tissue-engineered constructs remain to be clarified. In the present study, we syngenically transplanted tissue-engineered cartilage constructs consisting of C57BL/6 mice auricular chondrocytes and poly-l-lactic acid scaffolds (MW:200,000) into EGFP transgenic mice of C57BL/6 background, and evaluated the response by the localization of donor-derived and host-derived cells, the latter of which were distinguished by the presence of EGFP. While donor-derived cells constituted the areas of regenerated cartilage, host-derived cells were increased in number for the initial two weeks, and then decreased and excluded to non-cartilage areas thereafter. Furthermore, EGFP positivity was mostly co-localized with that of F4/80, suggesting most of the host-derived cells in the tissue-engineered constructs could be macrophages. Immunohistochemical staining of the tissue-engineered cartilage constructs revealed expression of factors related to immune privilege in chondrocytes, such as macrophage migration inhibitory factor (MIF), fas ligand (FasL) and others. Co-culture of chondrocytes and macrophages in vitro increased the expression of MIF and FasL in the chondrocytes, suggesting that chondrocytes in tissue-engineered cartilage constructs could regulate the actions of host-derived macrophages by expressing factors related to immune privilege.

Evaluation of irritancy and sensitization potential of metalworking fluid mixtures and components.

There are approximately 1.2 million workers exposed to metalworking fluids (MWF), which are used to reduce the heat and friction associated with industrial machining and grinding operations. Irritancy and sensitization potential of 9 National Toxicology Program (NTP) nominated MWFs (TRIM 229, TRIM VX, TRIM SC210, CIMTECH 310, CIMPERIAL 1070, CIMSTAR 3800, SYNTILO 1023, SUPEREDGE 6768, and CLEAREDGE 6584) were examined in a combined local lymph node assay (LLNA). BALB/c mice were dermally exposed to each MWF at concentrations up to 50%. Significant irritation was observed after dermal exposure to all MWFs except CIMTECH 310 and SYNTILO 1023. Of the 9 MWFs, 6 induced greater than a 3-fold increase in lymphocyte proliferation and 7 tested positive in the irritancy assay. TRIM VX yielded the lowest EC3 value (6.9%) with respect to lymphocyte proliferation. Chemical components of TRIM VX identified using HPLC were screened for sensitization potential using structural activity relationship (SAR) modeling and the LLNA. TOPKAT predicted triethanolamine (TEA) as a sensitizer while Derek for Windows predicted only 4-chloro-3-methylphenol (CMP) to be positive for sensitization. When tested in the LLNA only CMP (EC3 = 11.6%) and oleic acid (OA) (EC3 = 29.7%) were identified as sensitizers. Exposure to all tested TRIM VX components resulted in statistically significant irritation. An additive proliferative response was observed when mixtures of the two identified sensitizing TRIM VX components, OA and CMP, were tested in the LLNA. This is one explanation of why the EC3 value of TRIM VX, with respect to lymphocyte proliferation, is lower than those assigned to its sensitizing components.

Modification of the picryl chloride-induced allergic dermatitis model in mouse ear lobes by 12-O-tetradecanoylphorbol 13-acetate, and analysis of the role of histamine in the modified model.

BACKGROUND: In atopic dermatitis, inflammation induced by antigen-nonspecific stimuli further enhances the allergic inflammation. However, there is no experimental model in which allergic dermatitis is evoked where the inflammation has been induced by antigen-nonspecific stimuli. Here, we established a novel dermatitis model in mice and analyzed the role of histamine. METHODS: After sensitization with picryl chloride (PiCl) by painting on ear lobes of cyclophosphamide-treated mice, 12-O-tetradecanoylphorbol 13-acetate (TPA) was painted twice at the same site, and then allergic inflammation was induced by painting PiCl. Histamine antagonists and cyclosporine A (CsA) were administered intravenously. RESULTS: The application of TPA shifted the PiCl-induced allergic inflammation from a delayed-type response to a biphasic response, increased the infiltration of eosinophils and mast cells at the inflammatory site, shifted the cytokine milieu from Th1 to Th2 and induced the expression of thymic stromal lymphopoietin in the ear lobes. The PiCl-induced increase in the thickness of the ear lobe in the immediate phase was suppressed by the H1 antagonist pyrilamine. In contrast, the increase in the swelling in the late phase and the infiltration of eosinophils were suppressed by the H3/H4 antagonist thioperamide. The inhibitory effect of the combined treatment with pyrilamine and thioperamide on the TPA-modified contact dermatitis was as potent as that of CsA. CONCLUSION: Induction of the antigen-nonspecific inflammation by TPA enhanced the PiCl-induced allergic inflammation. Histamine plays significant roles in the early-phase swelling via H1 receptors, and the late-phase swelling via H3/H4 receptors in this TPA-modified allergic dermatitis model.

Nanoemulsions of an anti-oxidant synergy formulation containing gamma tocopherol have enhanced bioavailability and anti-inflammatory properties.

The present study investigated whether MicroFluidizer Processor-based nanoemulsions of an antioxidant synergy formulation (ASF), containing delta, alpha and gamma tocopherol influenced inflammation and bioavailability in CD-1 mice. Croton oil was applied to all animals' right ear lobe to induce inflammation. Auricular thickness was measured after 2 and 6h after the various treatments. The animal plasma and ear lobes were collected and frozen for bioavailability and cytokine analyses. The ASF nanoemulsions of alpha, delta, or gamma tocopherol significantly reduced auricular thickness compared to control (57, -57, and -71%, respectively) and blank nanoemulsion (-50, -50, -67%, respectively). Relative to the suspensions of ASF, only the nanoemulsion of ASF containing gamma tocopherol significantly reduced auricular thickness (-60%), whereas the 40% reduction with nanoemulsions of delta tocopherol compared to suspension was not statistically significant. Auricular concentrations of cytokines TNF-alpha and IL-1 alpha were significantly reduced in mice treated only with ASF nanoemulsions of gamma tocopherol compared to control (-53, -46%, respectively) and blank nanoemulsion (-52, -46%, respectively). Auricular thickness was significantly associated with tissue TNF-alpha (r=0.539, p<0.001) and IL-1 alpha concentrations (r=0.404, p=0.01). Bioavailability for gamma and delta was dramatically enhanced (2.2- and 2.4-folds) with the nanoemulsion compared to suspensions. Only the plasma gamma tocopherol concentration was significantly associated with auricular thickness (r=-0.643, P=0.001). In conclusion, nanoemulsions of ASF containing gamma, alpha, and delta tocopherol, have enhanced anti-inflammatory properties and increased bioavailability, with gamma tocopherol, in particular compared to their suspensions.

Sex-specific effects of glucose deprivation on cell-mediated immunity and reproduction in Siberian hamsters (Phodopus sungorus).

In most species, sexes differ in levels of parasitism. These differences have traditionally been believed to be static, but a capacity for adjusting anti-parasite investments would allow sexes to allocate resources adaptively contingent on environmental conditions. During stressful periods, such as a food shortage, allocation decisions would be mandated in males and females, but the biasing of resources may differ depending on the value of various physiological alternatives to the fitness of each sex. To determine whether sexes sacrifice immune or reproductive capacity when stressed, male and female Siberian hamsters (Phodopus sungorus) were pharmacologically deprived of glucose. Glucose deprivation was expected to compromise immune activity (delayed-type hypersensitivity) more than reproductive capacity in males because male fitness is limited by reproductive opportunities. The opposite was predicted for females because of the greater value of surviving to breed in favorable conditions. Contrary to expectations, glucoprivation compromised immune activity in female, but not male, hamsters. Conversely, glucoprivation reduced male, but not female, reproductive organ masses. These results may reflect the adjustments made by wild hamsters during food shortages, or they may be influenced by the study design; neither sex was permitted to incur other behavioral and physiological costs, such as lactation and parental care. Regardless, our results indicate that sex differences in parasitism are likely to be plastic in many circumstances, but further work in free-living animals is critical to ascertain whether results of the present study are naturally representative.

[Effects of berberine on DNFB-induced delayed type hypersensitivity in mice].

AIM: To investigate the effect of berberine (Ber) on DNFB-induced delayed type hypersensitivity (DTH) and explore the mechanism of the immunosuppression effect of berberine. METHODS: BALB/c mice were divided into three groups: control group, DTH group, and Ber-treated DTH group. The DTH group was sensitized to DNFB by painting the shaved abdomen with DNFB dissolved in propanone/olive oil. The Ber-treated DTH group was injected (i.p.) with berberine daily for 7 consecutive days, with the total dose of 30 mg/kg. The control group was treated with the solvent without DNFB. All mice were then challenged by painting 10 microL of 2 g/L DNFB on the left ears. The weight of right and left ears was measured at 48 h after challenge. Histological changes of mouse auricles were observed under light microscopy. Violet crystal staining was used to determine the effect of Ber on adhesion of mouse splenic lymphocytes to extracellular matrix (ECM). Annexin-V staining was used to detect the apoptotic rate of the lymphocytes in the lymph nodes. RESULTS: The results of ear weighting showed that Ber significantly inhibited DTH reaction (P<0.05). Histological observation indicated Ber reduced markedly the infiltration of lymphocytes. The adhesion of T lymphocytes to ECM was also notably decreased by Ber treatment (P<0.05). There was no obvious difference in apoptotic rates of the lymphocytes among Ber-treated DTH group, DTH group and control group (P>0.05). CONCLUSION: Ber can significantly inhibit the DNFB-induced DTH in mice. The reduced adhesion between mouse T lymphocytes and ECM may be one of the mechanisms of suppression of DTH by Ber.