This work was made possible by the support&#160; of Dr. Douglas Stairs, Marianne Klinger , Ann Benko, the Division of Rheumatology/Department of Medicine and the Molecular and Histopathologic Core Laboratory, Penn State University College of Medicine, Hershey PA.

All methods described were approved by the Penn State Institutional Review Board.
1. Histopathological Preparation, and Processing After Obtaining Temporal Artery Biopsy
NOTE:&#160;The temporal artery biopsy (TAB) is performed under local anesthesia and sterile conditions by a certified surgeon. After surgically obtaining a 3 cm arterial section on the more affected side, the specimen is fixed in formalin immediately for 24 h, divided into 3 to 4 mm s...

<ol>
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GCA is the most common vasculitis in adults, and its pathologic hallmark exemplifies a potent combination of T helper lymphocytes and activated macrophages that can also be found in other granulomatous diseases or vasculopathies like sarcoidosis and granulomatous polyangiitis2,3. In GCA, the temporal artery provides a good representation of a medium-to-large sized artery and the TA biopsy gives a sizeable sample that can be stored in paraffin blocks for years, th...

Giant cell arteritis (GCA) is an inflammatory disease of medium-to-large arteries, targeting the aorta and its branches and affecting older adults. It presents with mild to severe ischemic complications such as headaches, jaw pain, vision loss, stroke and tissue gangrene. The diagnosis is confirmed by high inflammatory markers like erythrocyte sedimentation rate (ESR) and a distinct histopathologic pattern on the temporal artery biopsy (TAB)1. GCA is the most common adult vasculitis, and the accessibility of the temporal artery obtained for diagnosis presents an advantage over other vasculopathies, thus enabling one to study its pathogenesis more readily. The typical findings on TAB include an infiltrate of macrophages/histiocytes and T lymphocytes found across all vascular layers of the tunica intima, media, and adventitia with concurrent destruction of the elastic lamina that normally separates these compartments2. The current evidence demonstrates that GCA involves an unknown antigen that activates dendritic cells in the vascular adventitia, followed by the recruitment of helper T (Th) cells, specifically Th1 and Th17 subtypes which secrete interleukin-17 and interferon-gamma (IFG) respectively. IFG then recruits and activates macrophages to produce cytokines and proteases. These include pro-inflammatory cytokines; including IL-12, which reciprocally activates Th1 cells thus providing a positive feedback loop; tumor necrosis factor&#160;and interleukin-6, which cause arthritis and fevers and metalloproteases that damage the elastic lamina. Glucocorticoids (GC), which constitute the standard chronic therapy, partially control the cytokine pathways by attenuating the Th17/IL-17 but not the Th1/IFG arm of the immune response3,4. Unfortunately, discontinuation of GC results in disease relapse. As an alternative modality, methotrexate has been repurposed for GCA treatment, but the desired clinical endpoints were not consistently achieved although more sensitive biomarkers have not been utilized for therapeutic monitoring5,6. In 2017, the interleukin 6 blocker, tocilizumab, was approved for the treatment of GCA as it effectively demonstrated disease control and steroid sparing effects7,8.
To date, there are no good biomarkers for GCA. As a result, it is difficult to monitor disease activity and titrate doses of available drugs to avoid adverse effects and reduce the burden of cost to society. Thus, it is imperative to look for a candidate biomarker that correlates with disease activity, provide novel insights on pathogenesis and aid in therapeutic decisions.
The dysregulation of macrophage activation is a critical factor in GCA pathogenesis. Thus, an effective means of treating GCA may be the selective targeting of activated macrophages. The folate receptor beta (FRB) is a glycosyl-phosphatidylinositol glycoprotein that is anchored on the cell membrane expressed in normal myelomonocytic cells, myeloid leukemia cells and activated macrophages. Its ligand, folic acid, is an essential vitamin in its reduced form, which enables cellular DNA synthesis, methylation and repair9. FRB expression is induced in autoimmune disease and during carcinogenesis. Its expression is restricted to the surface of monocytes or pro-inflammatory M1 macrophages in rheumatoid arthritis, or to anti-inflammatory M2 macrophages in solid organ and myeloid malignancies10,11,12,13. In addition to its potential role as a biomarker of activated macrophages, the FRB can enable selective drug transport thru a multi-step process that starts with the receptor binding to folic acid, antifolates or folate-conjugated drugs at the cell surface, followed by internalization, release and eventual delivery of desired drug to the internal cell machinery12,14,15,16. In contrast to FRB expressed in cancer cells and activated macrophages, FRB expressed in normal hematopoietic cells is unable to bind folates thereby ensuring that FRB/folate-mediated drug delivery is limited to FRB-positive inflammatory or cancer cells.
In our previous study, we demonstrated for the first time that FRB is expressed in GCA macrophages and its expression correlated with CD68+ and endothelin receptor beta macrophages, which contribute to GCA pathogenesis17. In this report, we describe the histopathological and immunohistochemical methods used to assess the FRB macrophage, and analyzed the total lymphocyte and macrophage counts to gain insight into the FRB&#39;s position in the GCA vascular landscape.

<table>
	<tbody>
		<tr>
			<td>Microtome</td>
			<td>Reichert-Jung</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>plain and frosted microscope slides and cover slips</td>
			<td>Fisher Scientific&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical rack</td>
			<td>Fisher Scientific&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Olympus BX60&#160; microscope</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone in distilled water</td>
			<td></td>
			<td></td>
			<td>concentrations 100%, 90%, 70%</td>
		</tr>
		<tr>
			<td>Tris-buffered saline solution (TBS)&#160;</td>
			<td>Dako Wash BufferS3006</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3% hydrogen peroxide in TBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Diaminobenzidine (DAB)</td>
			<td>Sigma Fast Tablet set</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemical Permount&#160; Mounting Medium</td>
			<td>Fisher Scientific&#160;</td>
			<td>SP-15-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Harleco Gill&#39;s III Hematoxylin</td>
			<td>Fiisher Scientific 23-750-019</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Harleco Eosin Y 1% Alcoholic Stock Solution</td>
			<td>Fisher Scientific&#160;</td>
			<td>23-749-977</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM citric acid monohydrate (pH 6.0)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polyclonal rabbit anti-folate receptor beta&#160;</td>
			<td>Manohar Ratnam</td>
			<td></td>
			<td>optimized at 1:800</td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-human CD68</td>
			<td>Dako&#160;</td>
			<td>M071801</td>
			<td>optimized at 1:200</td>
		</tr>
		<tr>
			<td>Polyclonal rabbit anti-CD3</td>
			<td>Agilent Dako</td>
			<td>A0452</td>
			<td>optimized at 1:100</td>
		</tr>
		<tr>
			<td>Polymer goat anti- rabbit/mouse secondary antibody</td>
			<td>Dako</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Placental tissue</td>
			<td></td>
			<td></td>
			<td>&#160;for FRB positive control</td>
		</tr>
	</tbody>
</table>

an immunohistopathologic study to profile the folate receptor beta macrophage and vascular immune microenvironment in giant cell arteritis

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis and occlusive symptoms of headaches, stroke or vision loss. Macrophages and T-helper lymphocytes infiltrate the vascular wall and produce a pro-inflammatory response that lead to vessel damage and ischemia. To date, there is no GCA biomarker that can monitor disease activity and guide therapeutic response.
Folate receptor beta (FRB) is a glycosylphosphatidylinositol protein that is anchored on cell membranes and normally expressed in the myelomonocytic lineage and in the majority of myeloid leukemia cells as well as in tumor and rheumatoid synovial macrophages, where its expression correlates with disease severity. The ability of FRB to bind folate compounds, folic acid-conjugates and antifolate drugs has made it a druggable target in cancer and inflammatory disease research. This report describes the histopathologic and immunohistochemical methods used to assess expression and distribution of FRB in relation to GCAimmunopathology.
Formalin-fixed and paraffin-embedded temporal artery biopsies from GCA and normal controls were stained with Hematoxylin and Eosin to review tissue histology and identify pathognomonic features.Immunohistochemistry was used to detect FRB, CD68 and CD3 expression. A microscopic analysis was performed to quantify the number of positively stained cells on 10 selected high-power-field sections and their respective locations in the arterial wall.
Lymphohistiocytic (LH) inflammation accompanied by intimal hyperplasia and disrupted elastic lamina was seen in GCA with none found in controls. The LH infiltrate was composed of approximately 60% lymphocytes and 40% macrophages. FRB expression was restricted to macrophages, comprising 31% of the total CD68+ macrophage population and localized to the media and adventitia. No FRB was seen in controls.
This protocol demonstrated a distinct numerical and spatial pattern of the FRB macrophage relative to the vascular immune microenvironment in GCA.

Histopathologic findings
The H&#38;E stains in normal specimens demonstrated normal arterial anatomy with endothelial cells in the tunica intima, smooth muscle cells in the tunica media, and a heterogenous collagen matrix which include fibroblasts and feeder vessels called vasa vasorum in the tunica adventitia. There is no inflammatory infiltrate identified. GCA positive temporal arteries demonstrated moderate t...

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An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis

The protocol illustrates the use of histopathologic examination and immunohistochemistry to profile the folate receptor beta macrophage and its relationship with the total immune cell infiltrate in temporal artery biopsies in giant cell arteritis.

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis ...

This immunohistochemical and histopathologic protocol can be used to examine folate receptor beta, which is a candidate protein with potential prognostic and therapeutic value to the vascular microenvironment in giant cell arteritis. This protocol is time-tested and widely employed and can be used to explore the folate receptor beta attributes in temporal artery tissues, even after long-term storage. To acquire tissue sections, use a microtome to slice four to five micrometer thick sections of paraffin-embedded temporal arteries, floating each section in a 40 degree Celsius water bath as they are obtained, to remove wrinkles.Use glass microscope slides to capture three sections per slide, and warm the slides on a warming plate, in a 60 degree Celsius oven for 60 minutes. When the sections have adhered to the slides, place the slides into a vertical rack for drying at 37 degrees celsius for 24 hours. The next day, transfer the slides to a slide container for storage at four degrees Celsius for at least 12 hours.Next, load slides into a slide rack and hydrate the tissues with two five-minute, 100%xylene immersions and a descending, five-minute per concentration ethanol immersion series, with 10 seconds of agitation every 30 seconds. After the 70%ethanol immersion, rinse the slides two times in double-distilled water for five minutes, with 10 seconds of agitation every 30 seconds. For antigen retrieval, transfer the rack into a glass container with 200 microliters of 95 degrees Celsius, 10 millimolar per liter citrate buffer for 30 minutes.At the end of the incubation, allow the slides to cool to room temperature for 20 minutes before rinsing with running water for five minutes. To remove the endogenous peroxidase activity, first use a hydrophobic marker to draw a circle around each tissue sample, before incubating the samples in 200 microliters of 3%hydrogen peroxide for 10 minutes, followed by 3 washes in 200 micoliters of tris-buffered saline solution, or TBSS, per wash. After the last wash, dab each slide gently to remove any excess buffer and add 200 microliters of the primary antibody cocktail of interest and a cover slip to each slide for a one-hour incubation in a humid chamber at room temperature.At the end of the incubation, discard the cover slips and rinse the slides with three two-minute washes in fresh TBSS. After removing the excess buffer, add 200 microliters of an appropriate biotinylated secondary antibody solution and a cover slip to each slide for a 45-minute incubation in the humid chamber at room temperature. After discarding the cover slips, rinse the slides with TBSS as demonstrated.Remove any excess buffer. And label the sections with 200 microliters of DAB substrate buffer for 10 minutes at room temperature. At the end of the incubation, wash the slides with TBSS and running tap water for 10 minutes, before counterstaining with hematoxylin for three minutes.Then, add 70 microliters of an appropriate mounting medium to each slide and slowly tip a glass cover strip onto the mounting medium to avoid creating bubbles as the cover slip is lowered into place. For histopathologic analysis, place the slide onto the stage of a light microscope and assess the vascular architecture of the first tissue section. Then, quantify the number of macrophages relative to the number of lymphocytes per section.For immunohistochemical analysis, examine the folate receptor beta staining pattern on each tissue section and quantify the number of folate receptor beta positive, CD68 positive, and CD3 positive cells in 10 randomly selected high power fields per section. Hematoxylin and eosin staining in normal specimens reveals a normal arterial anatomy, with endothelial cells in the tunica intima, smooth muscle cells in the tunica media, and a heterogeneous collagen matrix, that include fibroblasts and feeder vessels called vasa vasorum in the tunica adventitia. Giant cell arteritis positive temporal arteries demonstrate a moderate to severe inflammation, with aggregates and macrophages, or histiocytes, lymphocytes, occasional plasma cells, and multi-nucleated giant cells.Luminal narrowing is also noted, and accompanied by intimal thickening and a 90%disruption of the internal elastic lamina. CD3 positive lymphocytes and CD68 positive macrophages are also present in all giant cell arteritis positive specimens, with similar staining patterns observed in all of the biopsies. Staining for folate receptor beta is restricted to macrophages and multi-nucleated giant cells that preferentially localize to the adventitia and media.Notably, folate receptor beta comprised approximately 30%of the total macrophages. The temporal artery biopsy processing, cutting, and dewaxing require careful dexteriety in order to perform subsequent steps. The slide interpretation requires the expertise of an experienced cardiovascular pathologist.Remind the viewers to use contact and respiratory precautions, such as gloves and ventilated hoods, when handling specimens and the reagents.

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Immunology and Infection

7.0K visualizações.  Penn State University. Este protocolo imunohistoquímico e histopatológico pode ser usado para examinar o receptor beta, que é uma proteína candidata com potencial valor prognóstico e terapêutico ao microambiente vascular em arterite celular gigante. Este protocolo é testado por tempo e amplamente empregado e pode ser usado para explorar os atributos beta do receptor de folato em tecidos da artéria temporal, mesmo após o armazenamento a longo prazo. Para adquirir seções de tecido, use um microtome para co...