Research Techniques Made Simple: IMMUNOFLUORESCENCE Q&A

For details, we refer you to the article “Research Techniques Made Simple: Immunofluorescence Techniques” by Ian D. Odell1, MD, PhD and Deborah Cook2, MD

Departments of Medicine1 and Pathology2 University of Vermont College of Medicine, Burlington, VT

Questions

  1. What is the purpose of Michel’s solution?
    1. Fix the tissue before detection
    2. Cross link cellular components to retain the integrity of cellular structures
    3. Disrupt plasma membranes to give antibodies access to their target antigens
    4. Precipitate the immune complexes to preserve antigenicity

 

  1. How many additional antibodies are required to detect autoimmune complexes?
    1. 1
    2. 2
    3. 3
    4. 4

 

  1. Which of the following techniques is more sensitive than immunofluorescence for the diagnosis of some autoimmune bullous diseases?
    1. Light microscopy
    2. ELISA
    3. Dermatoscope
    4. Western blot

Answers

1. D

2. A

3. B

Editors’ Picks from Experimental Dermatology (December 2012)

New insights into the anti-inflammatory, anti-tumorigenic and anti-angiogenic properties of PPARβ/δ agonists: Targeting cathepsin B expression and function

 

Previous studies indicate that peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a promising drug target involved in the serum increase of high-density lipoprotein, the decrease of triglycerids, the promotion of terminal differentiation, and anti-inflammatory activities. In addition, there are first studies of anti-carcinogenic and anti-angiogenic effects. The underlying mechanisms of PPARβ/δ agonist mediated action still had to be elucidated. Reichenbach and colleagues (2012) recently found that PPARβ/δ agonists suppress cathepsin B expression in human endothelial cells in a posttranslational manner. The expression of other cathepsins seemed to be unaffected. Transcriptional or posttranscriptional mechanisms as 3´-UTR- or miRNA-dependent mechanisms could be excluded. Cathepsin B is an important protease known to be involved in the process of angiogenesis, facilitating capillary tube formation, as well as in tumor metastasis. Therefore its suppression by PPARβ/δ agonists, as described by Reichenbach and colleagues, offers new support of the concept of their anti-inflammatory and anti-tumorigenic value.

Selected by R. Ludwig, Lübeck, Germany

Ligand activation of peroxisome proliferator-activated receptor delta suppresses cathepsin B expression in human endothelial cells in a posttranslational manner.  Reichenbach G, Starzinski-Powitz A, Doll M, Hrgovic I, Maria Valesky E, Kippenberger S, Bernd A, Kaufmann R, Meissner M.  Exp Dermatol. 2012 Oct;21(10):751-7. doi: 10.1111/exd.12002.

 

 

Protein kinase A activation suppresses sonic hedgehog signalling and basal cell carcinoma

Activation of the sonic hedgehog (Shh) pathway is the underlying mechanism for basal cell carcinomas. In this pathway the membrane Shh receptor patched suppresses smoothened (Smo) until the suppression is relieved by binding of Shh to patched. Smo, when relieved of this suppression, in turn activates the transcription factors gli 1 and 2, which then enter the nucleus and turn on genes leading to BCC. Drugs to block Smo are currently on the market and/or under development, but their toxicity is substantial and their use leads to resistance based on mutations in Smo.  So other treatment modalities would be welcome.   E. Makinodan and A.G. Marneros (2012) used an agonist of protein kinase A, forskolin, which has been shown to phosphorylate gli 1 and 2, leading to their proteosomal degradation, to demonstrate in vivo and in vitro that forskolin could block BCC development. In particular, for the in vitro studies they used a cell line, SmoA1-LIGHT2, which contains a gli dependent luciferase and a constitutively active Smo construct to demonstrate that forskolin blocked gli 1 activation of the luciferase construct. In the in vivo study they used a mouse, R26SmoM2, that when bred to a mouse with tamoxifen regulated (ERT2) K14-cre recombinase expressed a constitutively active Smo, to demonstrate that forskolin could prevent tumor formation on the ears of these mice. Whether forskolin or similar agents can be used clinically in a topical fashion to prevent or reverse BCC progression remains to be seen.

Selected by D. Bikle, San Francisco, CA, USA

Protein kinase A activation inhibits oncogenic Sonic hedgehog signalling and suppresses basal cell carcinoma of the skin. Makinodan E, Marneros AG.  Exp Dermatol. 2012 Nov;21(11):847-52. doi: 10.1111/exd.12016.

 

 

Spotlight on tissue engineering to manage chronic wounds

Restoration of skin integrity in impaired healing conditions associated with aging, metabolic, or vascular disease remains an unresolved and challenging task in medicine. Wong and Gurtner provide a comprehensive and concise viewpoint on recent therapeutic developments in biotechnology and point out exciting perspectives for future research in this field (2012). The authors outline the synergistic efforts of biomedical engineers, material scientists, and cell biologists during the past decade that yielded promising therapies to manage difficult-to-treat wounds. Most importantly, rigorous basic science and clinical studies are now required to validate these novel approaches. Furthermore, recent advances in the biology of tissue repair stipulate that in addition to the inflammation-based paradigm of wound healing pathology, the role of stem cells, matrix components, and other – potentially yet unknown – molecular pathways must be further explored in current and evolving therapeutic concepts for chronic wounds.

Selected by S.A. Eming, Cologne, Germany

Tissue engineering for the management of chronic wounds: current concepts and future perspectives. Wong VW, Gurtner GC.  Exp Dermatol. 2012 Oct;21(10):729-34. doi: 10.1111/j.1600-0625.2012.01542.x.

 

 

Rosacea: inflammatory reaction of the skin immune system (SIS) to outside agents?

Christiane Casas and colleagues address the topic of Demodex folliculorum density and inflammatory protein expression in the facial skin of rosacea patients (2012). The authors report a multicentre, cross-sectional, prospective study including 50 patients with with well-defined types of facial rosacea, and 48 age- and sex-matched healthy volunteers. Patients with isolated rhinophyma, isolated ocular rosacea, granulomatous rosacea, fulminant rosacea, or steroid-induced rosacea were excluded, as were patients using antiparasitics, antibiotics active against Bacillus oleronius, or local antiseptics. Facial skin scrapings (micro-abrasions) and swabs were studied for D. folliculorum density as well as for a number of inflammation markers.

The results point to neutrophil activation induced by bacterial proteins associated with Demodex in rosacea patients. Moreover, they suggest that the NLRP3 inflammasome might be a new factor in the pathogenesis of rosacea by promoting excessive secretion of IL-1b. Furthermore, IL-18 underexpression, together with that of the ASC adaptor, could be responsible for a loss of microbial homoeostasis.

There is no doubt that there is inflammation in patients suffering from rosacea. Patients feel the burning accompanying that inflammatory response. A role for the hair follicle mite, Demodex folliculorum, has long been suspected in this inflammation. Recent findings indicate that not Demodex itself is the culprit, but the Bacillus oleronius that these mites can harbour (Jianjing L et al, 2010). Therefore, the authors excluded patients being treated with antibiotics that might have eradicated these micro-organisms.

While this work brings us closer to the goal of fully understanding the mechanisms of this sometimes devastating disease, several questions arise: Is it primarily dysfunction of the innate part of the skin immune system (SIS) that enables otherwise harmless inhabitants of the hair follicles to induce inflammation? Is a subtype of Bacillus oleronius responsible for that inflammation? Or is it, perhaps, a combination of both? Why is rosacea more common in women, peaking around age 40? Why is it so often a temporary ailment that can resolve spontaneously? Quite possibly, we have as yet only scratched the surface of the complex interplay between the skin microbiome and the SIS in rosacea.

Selected by J.D. Bos, Amsterdam, the Netherlands

Quantification of Demodex folliculorum by PCR in rosacea and its relationship to skin innate immune activation. Casas C, Paul C, Lahfa M, Livideanu B, Lejeune O, Alvarez-Georges S, Saint-Martory C, Degouy A, Mengeaud V, Ginisty H, Durbise E, Schmitt AM, Redoulès D.  Exp Dermatol. 2012 Dec;21(12):906-10. doi: 10.1111/exd.12030

Correlation between ocular Demodex infestation and serum immunoreactivity to Bacillus proteins in patients with Facial rosacea.  Li J, O’Reilly N, Sheha H, Katz R, Raju VK, Kavanagh K, Tseng SC.  Ophthalmology. 2010 May;117(5):870-877.e1.

 

 

Investigating stratum corneum lipids by electron spectroscopic analysis 

The lipids of the stratum corneum comprise the main components of the hydrophobic human skin barrier regulating water homoeostasis. Recently, the notion that mutations in the filaggrin gene result in ichthyosis vulgaris and are associated with atopic eczema have raised interest in the normal human skin lipid barrier, and in how skin lipids change in diseased skin.

Using the latest X-ray photoelectron spectroscopy technology, Marschewski et al. (2012) found that the human lipid skin barrier consists of 84.4% carbon, 10.8% oxygen and 4.8% nitrogen. In addition, they identified for the first time a fundamental and reproducible influence of cold atmospheric plasma treatment on the lipid barrier system of human skin, as can be especially derived from the clear structural changes in the oxygen detailed spectra.

Their experimental setting and results pave the way for more detailed fundamental analyses of the effects of plasma treatment on the lipid barrier of human skin, thus deciphering effects on each of the three main lipid components of that barrier, namely ceramides, cholesterol, and long-chain free fatty acids.

Selected by K. Kabashima, Kyoto, Japan

Electron spectroscopic analysis of the human lipid skin barrier: cold atmospheric plasma-induced changes in lipid composition. Marschewski M, Hirschberg J, Omairi T, Höfft O, Viöl W, Emmert S, Maus-Friedrichs W.  Exp Dermatol. 2012 Dec;21(12):921-5. doi: 10.1111/exd.12043.

Dealing with Complexity

Image credit: istockphoto: 19814069, by Ma_co.

 

Health depends on the proper functioning of complex biological systems responsive to multiple environmental effects, and understanding disease depends on analyzing how complex systems go awry. In many circumstances we are beyond the simple analysis of component “A” goes up and then component ”B” goes down. Single lines on gel blots will become relics  of our past. Some  may wish that  they lived and performed science in the era of gold standard Koch postulates, but that is not our time. As mentioned in previous blogs we now require more complex mathematical analysis and constructs that either we learn to perform ourselves or for which we seek knowledgeable and state-of-the-art collaborators.  Three recent methodology articles bear reviewing first by yourself and them with a wise colleague.

In a review in Science (Brennan et al, 2012), signaling and heterogeneity in cell populations and the variability (noise) seen in systems is considered from the standpoint of basic information theory. Theories on how cells or groups of cells get optimal information at the minimum metabolic cost are considered. No equations in this one, so breathe easily.

In the same issue, Scheffer et al (2012) consider critical transitions in systems and how heterogeneous systems may be considerably robust and resilient — and adaptive to multiple stresses. There are examples from chemistry and physiology to ecology.

Finally, the question of causality is approached — and avoiding the traps of  inferring causality from correlation (Sugihara et al, 2012).  This is a timely topic in these days of interpreting  the fall elections. Discussed in detail are the relationships between anchovy populations and sardine populations in  the Pacific ocean. These are questions that may not be approached through experiments.

I suggest that the first step is to develop seminar series that may be beyond the usual comfort levels of clinical or basic scientists so that these types of analyses can be explored with new groupings of smart folks. A modest idea.

 

REFERENCES

Brennan MD, Cheong R, Levchenko A, et al. How Information Theory Handles cell signaling and uncertainty Science 338:334-5, 2012

Scheffer M , Carpenter SR, Lenton TM, et al. Anticipating critical transitions. Science 338:344-8, 2012

Sugihara G , May R, Ye H, et al. Detecting Causality in Complex Ecosystems Science 338:496-500, 2012