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 sections, and embedded in paraffin, paying careful attention to the proper alignment of tissue in the block which is then&#160; stored at room temperature. Specimen selection is performed after verifying the medical records. The diagnosis is based on the American College of Rheumatology 1990 criteria which requires that subjects fulfill 3 of 5 domains: age &#62;50 years, new headache, temporal artery tenderness, an erythrocyte sedimentation rate &#62;50 mm/hour by Westergren method and an abnormal TAB.&#160;For safety, protective gloves must be worn at all times when handling the specimens, chemicals and antibodies.
<ol>
	<li>From the embedded blocks, cut 4-5 &#181;m thick sections using a microtome. and stain with Hematoxylin and Eosin (H&#38;E). Use a control section of selected normal tissue for quality assurance.</li>
	<li>Float cut sections on a warm water bath heated to 40 &#176;C to remove wrinkles and pick up with a coated glass microscopic slide.&#160; Place the glass slides on a warm plate in a 60 &#176;C oven for 60 minutes to facilitate drying and adherence of section to the slide.</li>
	<li>Place the glass slides on a warm plate in a 60 &#176;C oven for 60 minutes to facilitate drying and adherence of section to the slide.</li>
	<li>Mount 3 sections on microscope slides.</li>
	<li>Place slides in a vertical rack and dry overnight at 37 &#176;C for 24 h.</li>
	<li>Place slides in a container and store at 4 &#176;C.</li>
</ol>
2. Immunohistochemical Preparation, Dewaxing,&#160;Antigen Retrieval and Staining15,18,19,20
<ol>
	<li>Load slides into a slide rack. Run the rack through dishes as follows: Twice in 100% xylene for 5 minutes with 10 seconds of gentle agitation every 30 seconds, once in 100% ethanol with 10 seconds agitation, once in 90% ethanol with 10 seconds agitation, once in 70% ethanol with 10 seconds agitation, and twice in double distilled H2O with 10 seconds agitation.&#160; 
	Note: Handling of xylene and acetone should be done in ventilated hoods to avoid inhalation and respiratory toxicity.</li>
	<li>Retrieve the antigen by transferring the rack into a glass container with 200 mL of 10 mmol/L, pH 6.0 citrate buffer prewarmed to 95 &#176;C. Set the timer for 30 minutes in the water bath, then cool in room temperature for 20 minutes then rinse with running water for 5 minutes.</li>
	<li>Remove endogenous peroxidase activity by incubating in 200 mL of 3% hydrogen peroxide for 10 minutes. Wash slides 3 times in 200 &#181;L of Tris-buffered saline solution (TBSS).</li>
	<li>Remove the slides from the rack and dab each one gently to wipe excess buffer. For staining, add 200 &#181;L of the following primary antibodies, add cover slips on slides and incubate in a humid chamber for 1 hour at room temperature: 
	Anti-FRB antibody diluted 1:800 
	Monoclonal mouse anti-human CD68, 1:200 dilution 
	Polyclonal rabbit anti-CD3 1:100 dilution 
	Note: Formalin-fixed paraffin-embedded sections of placenta were also processed as outlined in Steps 2.1 to 2.9 and incubated with anti-FRB18 and used as positive control. Staining results were obtained by finding the optimal antibody concentration and was performed by titrating the antibody in double dilutions (e.g. 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600).</li>
	<li>Remove cover slip after incubation and discard. Rinse slides 3 times for 2 minutes each with TBSS, drain and wipe excess buffer.</li>
	<li>Add 200 &#181;L of secondary antibody solution containing biotinylated Anti-Rabbit/Mouse secondary antibody to react with unconjugated primary antibody bound to tissue antigen. Place coverslips on slides and incubate in a humid chamber for 45 minutes at room temperature.</li>
	<li>Remove cover slip after incubation and discard. Rinse slides 3 times for 2 minutes each with TBS, drain and wipe excess buffer.</li>
	<li>Add 200 &#181;L of diaminobenzidine (DAB)+substrate buffer (Imidazole HCl) -chromogen system and incubate for 10 minutes to visualize staining pattern. Wash with TBSS then in running tap water for 10 minutes.</li>
	<li>Counterstain with hematoxylin for 3 minutes.</li>
	<li>Mount the slides by applying 70 &#181;L of mounting medium to the surface of the slide. Slowly tip the coverslip onto the mounting medium and avoid creating bubbles as you lower it into place. Wait 24 hours to dry.&#160;</li>
</ol>
3. Histopathologic and Immunohistochemical (IHC) Analysis
Examine the H&#38;E and IHC stained sections from GCA positive and normal subjects using a light microscope.
<ol>
	<li>For the H&#38;E stained sections:
	<ol>
		<li>Assess the vascular architecture and identify endothelial cells in the tunica intima, smooth muscle cells in the tunica media, and fibroblasts and vasa vasorum in the tunica adventitia21,22.</li>
		<li>Identify GCA features, which include lymphocyte and macrophage/histiocyte infiltration, cellular hyperplasia, and internal elastic lamina degradation.</li>
		<li>Analyze the lymphohistiocytic infiltrate by identifying and quantifying the number of macrophages relative to the lymphocytes. Assess the extent of internal elastic lamina disruption and hyperplastic changes in resident cells and compare with controls.</li>
	</ol>
	</li>
	<li>For the IHC stained sections:
	<ol>
		<li>Examine the staining pattern of FRB, taking note of its expression by a particular type or types of cells and its distribution within the vascular layers and its relationship to the total immune infiltrate, the total macrophage marker, anti-CD68, and the pan lymphocyte marker anti CD3.</li>
		<li>Quantify the expression of FRB, CD68 and CD3 cells by counting the positively stained cells on 10 multiple randomly selected high power fields (hpf) and record the means. 
		NOTE: Identification of these normal and pathologic cells and structures were performed by a certified cardiovascular pathologist (J.M.) and rheumatologist (S.A.A.) and the results were recorded for all cases.</li>
		<li>Capture representative images using a digital camera. 
		NOTE: After these steps, remaining aliquots may be stored at -80 &#176;C.</li>
	</ol>
	</li>
</ol>

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunopathology+of+giant+cell+arteritis:+diagnostic+and+therapeutic+implications.">The immunopathology of giant cell arteritis: diagnostic and therapeutic implications.</a> Journal of Neuro-ophthalmology. 32 (3), 259-265 (2012).</li><li>Deng, J., Younge, B., Olshen, R., Goronzy, J., Weyand, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th17+and+Th1+T-cell+responses+in+giant+cell+arteritis.">Th17 and Th1 T-cell responses in giant cell arteritis.</a> Circulation. 121, 906-915 (2010).</li><li>Mahr, A. 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The authors have nothing to disclose.

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 ...

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Immunology and Infection

7.1K Views.  Penn State University. 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.

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

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident cells of star-like morphology and are located in the space of Disse between liver sinusoidal endothelial cells and hepatocytes1,2. Stellate cells are derived from bone marrow precursors and store up to 80% of the total body vitamin A1, 2. Upon activation, stellate cells differentiate into myofibroblasts to produce extracellular matrix, thus contributing to liver fibrosis3. Based on their ability to contract, myofibroblastic stellate cells can regulate the vascular tone associated with portal hypertension4. Recently, we demonstrated that hepatic stellate cells are potent antigen presenting cells and can activate NKT cells as well as conventional T lymphocytes5. 

 Here we present a method for the efficient preparation of hepatic stellate cells from mouse liver. Due to their perisinusoidal localization, the isolation of hepatic stellate cells is a multi-step process. In order to render stellate cells accessible to isolation from the space of Disse, mouse livers are perfused in situ with the digestive enzymes Pronase E and Collagenase P. Following perfusion, the liver tissue is subjected to additional enzymatic treatment with Pronase E and Collagenase P in vitro. Subsequently, the method takes advantage of the massive amount of vitamin A-storing lipid droplets in hepatic stellate cells. This feature allows the separation of stellate cells from other hepatic cell types by centrifugation on an 8% Nycodenz gradient. The protocol described here yields a highly pure and homogenous population of stellate cells. Purity of preparations can be assessed by staining for the marker molecule glial fibrillary acidic protein (GFAP), prior to analysis by fluorescence microscopy or flow cytometry. Further, light microscopy reveals the unique appearance of star-shaped hepatic stellate cells that harbor high amounts of lipid droplets. 


 Taken together, we present a detailed protocol for the efficient isolation of hepatic stellate cells, including representative images of their morphological appearance and GFAP expression that help to define the stellate cell entity.

Here we describe a method for the isolation of hepatic stellate cells from mouse liver. For stellate cell purification, mouse livers are digested in situ and in vitro by pronase-collagenase treatment prior to density gradient centrifugation. This technique yields highly pure hepatic stellate cells.

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.

This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens.

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for understanding the function of bacterial cells in the natural environment. Because of technological advances, various bacterial cell functions can be revealed at the single-cell level inside a confined space. GVs are spherical micro-sized compartments composed of amphiphilic lipid molecules and can hold various materials, including cells. In this study, a single bacterial cell was encapsulated into 10&#8211;30 &#956;m GVs by the droplet transfer method and the GVs containing bacterial cells were immobilized on a supported membrane on a glass substrate. Our method is useful for observing the real-time growth of single bacteria inside GVs. We cultured Escherichia coli (E. coli) cells as a model inside GVs, but this method can be adapted to other cell types. Our method can be used in the science and industrial fields of microbiology, biology, biotechnology, and synthetic biology.

We demonstrate single-cell culture of bacteria inside giant vesicles (GVs). GVs containing bacterial cells were prepared by the droplet transfer method and were immobilized on a supported membrane on a glass substrate for direct observation of bacterial growth. This approach may also be adaptable to other cells.