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Publications Reporting Intradermal Injection

Publication 1.

H.A. Lankes, C.N. Zanghi, K. Santos, C. Capella, C.M.P. Duke, S. Dewhurst, In vivo gene delivery and expression by bacteriophage lambda vectors , Journal of Applied Microbiology , 102 (2007) 1337–1349.

 

Aims: Bacteriophage vectors have potential as gene transfer and vaccine delivery vectors because of their low cost, safety and physical stability. However, little is known concerning phage-mediated gene transfer in mammalian hosts. We therefore performed experiments to examine phage-mediated gene transfer in vivo .
Methods and Results: Mice were inoculated with recombinant lambda phage containing a mammalian expression cassette encoding firefly luciferase (luc). Efficient, dose-dependent in vivo luc expression was detected, which peaked within 24 h of delivery and declined to undetectable levels within a week. Display of an integrin-binding peptide increased cellular internalization of phage in vitro and enhanced phage-mediated gene transfer in vivo . Finally, in vivo depletion of phagocytic cells using clodronate liposomes had only a minor effect on the efficiency of phage-mediated gene transfer.
Conclusions: Unmodified lambda phage particles are capable of transducing mammalian cells in vivo , and may be taken up – at least in part – by nonphagocytic mechanisms. Surface modifications that enhance phage uptake result in more efficient in vivo gene transfer.
Significance and Impact of the Study: These experiments shed light on the mechanisms involved in phage-mediated gene transfer in vivo , and suggest new approaches that may enhance the efficiency of this process.

Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
5 mg/ml 23.5 mg/ml EPC/Chol 86/14 MLV none

 

Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Systemic Dosing? Systemic Results
BALB/c mice 100 µl tail base intradermal phagocytes 200 µl i.p. F4/80+ splenocytes depleted

Notes

  1. Transfection initiated 48-hours post-clodronate liposome treatment, however intradermal phagocyte depletion was not confirmed. Most models require 3 or more days to achieve maximal depletion.

Results

  1. F4/80+ splenocytes reduced from 14.84% to 0.34% of total cells by FACS; intradermal phagocytes not assessed.
  2. Phage lambda-mediated gene-transfer is only modestly affected by presumed intradermal phagocyte depletion suggesting that phagocytosis is not the predominant mechanism of transfection.

 

Publication 2.

D.M. Catron, K.A. Pape, B.T. Fife, N. van Rooijen, M.K. Jenkins, A Protease-Dependent Mechanism for Initiating T-Dependent B Cell Responses to Large Particulate Antigens, The Journal of Immunology, 184 (2010) 3609–3617.

 

Ab production is critical for antimicrobial immunity, and the initial step in this process is the binding of Ag to the BCR. It has been shown that small soluble proteins can directly access the lymph node follicles to reach naive B cells, but virus particles must be translocated into follicles via subcapsular sinus macrophages. In this article, we explore how large particulate Ags generate humoral immune responses. Ag-specific follicular B cells rapidly acquired Ag, presented peptide:MHC class II ligands, and produced T-dependent Ab responses following s.c. injection of 1-µm, Ag-linked microspheres, despite the microspheres being confined to the subcapsular sinus. The mechanism of Ag acquisition did not require dendritic cells, subcapsular sinus macrophages, or B cell movement to the subcapsular sinus. Rather, B cell Ag acquisition was protease-dependent, suggesting that some protein Ags are cleaved from the surface of particles to directly initiate humoral immune responses.

Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
5 mg/ml 23.5 mg/ml EPC/Chol 86/14 MLV PBS

 

Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Systemic Dosing? Systemic Results
C57BL/6 mice, 6-8 wks 2 10 µl pinna MOMA-1+ macrophages no not evaluated

Notes

  1. MOMA-1+ macrophage depletion was visually determined by confocal microscopy of non-draining lymph nodes but not assigned numerical values.

Results

  1. Macrophage depletion did not inhibit or reduce antigen presentation in the lymph nodes.

 

Publication 3.

P.C. Cook, S.A. Aynsley, J.D. Turner, G.R. Jenkins, N. Van Rooijen, M. Leeto, F. Brombacher, A.P. Mountford, Multiple Helminth Infection of the Skin Causes Lymphocyte Hypo-Responsiveness Mediated by Th2 Conditioning of Dermal Myeloid Cells , PLoS Pathogens , 7 (2011) e1001323.

 

Infection of the mammalian host by schistosome larvae occurs via the skin, although nothing is known about the development of immune responses to multiple exposures of schistosome larvae, and/or their excretory/secretory (E/S) products. Here, we show that multiple (4x) exposures, prior to the onset of egg laying by adult worms, modulate the skin immune response and induce CD4+ cell hypo-responsiveness in the draining lymph node, and even modulate the formation of hepatic egg-induced granulomas. Compared to mice exposed to a single infection (1x), dermal cells from multiply infected mice (4x), were less able to support lymph node cell proliferation. Analysis of dermal cells showed that the most abundant in 4x mice were eosinophils (F4/80+MHC-II−), but they did not impact the ability of antigen presenting cells (APC) to support lymphocyte proliferation to parasite antigen in vitro . However, two other cell populations from the dermal site of infection appear to have a critical role. The first comprises arginase-1+, Ym-1+ alternatively activated macrophage-like cells, and the second are functionally compromised MHC-IIhi cells. Through the administration of exogenous IL-12 to multiply infected mice, we show that these suppressive myeloid cell phenotypes form as a consequence of events in the skin, most notably an enrichment of IL-4 and IL-13, likely resulting from an influx of RELMα-expressing eosinophils. We further illustrate that the development of these suppressive dermal cells is dependent upon IL-4Rα signalling. The development of immune hypo-responsiveness to schistosome larvae and their effect on the subsequent response to the immunopathogenic egg is important in appreciating how immune responses to helminth infections are modulated by repeated exposure to the infective early stages of development.

Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
5 mg/ml 23.5 mg/ml EPC/Chol 86/14 MLV PBS

 

Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Systemic Dosing? Systemic Results
C57BL/6 mice, female, 8-12 wks 2 ? µl pinna Arg-1+, Ym-1+ AAMΦ-like cells, functionally compromised MHC-II hi DC no not evaluated

Notes

  1. Clodronate or control liposomes (volume not specified) were administered intradermally 72 hrs prior to each of the first 3 infections. These mice were infected with 100 cecariae on days 0, 7, 14, and 21 followed by sacrifice on day 35 (total 4X infections). Given that over 2 weeks passed between the last clodronate liposome treatment and sacrifice, and that no systemic clodronate liposome treatment was given, it seems likely that the depletion may have been more extensive at earlier time points.

Results

  1. MHC-II lo alternatively activated macrophage-like (AAMΦ-like) cells  and MHC class II hi dendritic cells (DC) were reduced by ~60% in the skin.
  2. Cell depletion resulted in an increase in local skin-draining lymph node (sdLN) cell proliferation and interferon-γ.
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Publication 4.

N.L. Ward, C.M. Loyd, J.A. Wolfram, D. Diaconu, C.M. Michaels, T.S. McCormick, Depletion of antigen-presenting cells by clodronate liposomes reverses the psoriatic skin phenotype in KC-Tie2 mice , British Journal of Dermatology , 164 (2011) 750–758.

Background: There is ongoing debate regarding the initiation of psoriatic plaque as primarily arising from an anomaly in epidermal keratinocytes (KCs) or from abnormalities in immunocytes that secondarily activate otherwise normal KCs. In mice engineered to overexpress the angiopoietin receptor Tie2 in KCs, skin spontaneously develops the characteristic clinical, histological and immune cell phenotypes of psoriasis which can be reversed with either transgene repression or ciclosporin administration, suggesting key roles for both KCs and T cells in mediating the skin disease in this murine model.
Objectives: To determine if antigen-presenting cells (APCs) and macrophages alone are sufficient to sustain psoriasiform inflammation in the KC-Tie2 murine model of psoriasis.
Methods: Clodronate liposomes were intradermally injected into involved dorsal skin of KC-Tie2 or control animals once a week for 6 weeks and acanthosis, angiogenesis, immune cell infiltration and cytokine production were quantitated using immunohistochemistry and interactive image analyses, enzyme-linked immunosorbent assay (ELISAs) and quantitative real-time polymerase chain reaction (qRT-PCR).
Results: Clodronate liposome injection eliminated CD11c+, F4⁄80+ and CD11b+ cells in the skin and returned CD8+ T-cell numbers to control mouse levels. APC depletion in KC-Tie2 mouse skin resulted in resolution of the acanthotic skin phenotype, decreased dermal angiogenesis, and a return to control mouse levels for interleukin (IL)-1α, IL-6, IL-23 and tumour necrosis factor (TNF)-α expression and modest reductions in interferon-γ and IL-17.
Conclusions: These findings suggest a critical role for APCs and myeloid cell-derived IL-23 and TNF-α and underscore the importance of Th1 and Th17 T cells in maintaining the psoriasiform skin phenotype in the KC-Tie2 mouse model.

Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
5 mg/ml 23.5 mg/ml EPC/Chol 86/14 MLV PBS

 

Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Systemic Dosing? Systemic Results
KC-Tie2 mice, adult 2 200 µl 50 µl X 4 dorsal sites F4/80+ macrophages, CD11b+ myeloid cells, CD11c+ dendritic cells no not evaluated

Notes

  1. 50 µl of clodronate or control liposomes were injected intradermally into 4 dorsal sites (total dose = 200 µl) once per week X 6 weeks.
  2. Citation referencing injection method reported using subcutaneous injections rather than intradermal, therefore results may not be comparable.
  3. Depletion determined by counting cells in a defined field of immunohistochemically stained tissue.

Results

  1. Control (PBS) liposomes resulted in the level of APC increasing by about 2X above controls (disease-free animals), but clodronate liposomes reduced APC levels to control levels.

 

2 Base or background strain. Other variants, mutants or genetically-modified animals were also used in this study.

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