Grant Awarded in 2010
Flow Cytometric and Secretomic Analysis of LCH Lesions
Institute of Cellular Medicine – Newcastle upon Tyne UK
Date of Award
Amount of Award
The title of this project is “Flow Cytometric and Secretomic Analysis of LCH Lesions.” Langerhans cell histiocytosis (LCH) is a rare but sometimes fatal disorder of unknown cause. The lesions of LCH consist of abnormal ‘LCH cells’ together with other immune cells including T lymphocytes, macrophages and eosinophils. LCH cells are an important feature for making the diagnosis of LCH but it is likely that all the cells of the lesion work together to cause the disease. We are using two modern tools—flow cytometry and secretomics (proteomics of secreted proteins)—to look in greater detail at the cells and molecules of LCH.
Flow cytometry is a means of analyzing mixtures of cells in great detail from small amounts of tissue. It is ideal for analyzing LCH by describing what cells are present, how many of each type of cell exists, and what their activity or functional status shows. This will allow us to see if lesions from different sites in patients with low-risk or high-risk disease look different. In the future this should enable us to diagnose LCH more quickly and make a more accurate prognosis.
Another aim of the study is to see if LCH cells have a normal counterpart in the body. Although we think of LCH as derived from Langerhans cells, other populations of cells with some features more closely resembling LCH also exist. Our studies will test the closeness of this relationship.
Finally, we want to understand the signals that pass between cells in the lesion. To do this, we will put LCH biopsy material into in-vitro culture and then analyze the culture medium using sensitive assays for specific proteins (ELISA), and discovery tools for new disease markers (mass spectroscopy). Using these techniques, we hope to understand more about the abnormal signaling between cells to help understand the mechanism of the disease and to discover new markers that will allow us to monitor the disease using blood tests.
Twelve Month Report
The title of this project is Flow cytometric and secretomic analysis of LCH lesions. Through this work we aimed to achieve a more detailed understanding of the type of cells in LCH lesions, how they relate to normal healthy tissues and what causes them to form. We used flow cytometry, a method of analyzing many thousands of cells form small tissue samples to look at LCH lesions and normal tissues. We found:
- Flow cytometry can be used to make a rapid diagnosis of LCH within 24 hours of biopsy. This work will allow patients to be diagnosed more quickly and also gives us information about the type of cells in LCH lesions. We also discovered that most lesions have two types of LCH cells mixed together. We are currently working out the differences between theses two types and whether they come from different white blood cells. We will write a paper based on these results called ‘Rapid diagnosis of LCH by flow cytometry’
- We have extensively analysed normal human tissues to look for cells related to LCH cells that express Langerin. The reason for doing this was to give us a clue about why LCH lesions develop. We found a new type of human DC identified by high expression of CD141. In the mouse, this type of DC expresses Langerin, but not in human. Instead, in humans we found another CD1c+ DC that expresses Langerin. We think that this cell may be related to one of the LCH cells. These findings will be published in two papers: 1) ‘Human interstitial tissues contain cross-presenting CD141high DCs’; 2) ‘Interstitial dermal dendritic cells express langerin and are distinct from Langerhans cells: implications for the pathogenesis of LCH.’
- Looking for reasons why Langerin appears on some DCs, we developed an in vitro experiment in which Langerin appears on blood DCs. A really striking finding was that LCH patients express much more Langerin than healthy controls. This offers the hope that we could have an in vitro test for LCH. We need to expand this test to many more patients to determine how good it might be.
Overall we have achieved useful insights into the diagnosis or LCH and the reason it develops. We thank the HA for its generous support.
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