Lymph node

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Lymph node
Schematic of lymph node showing lymph sinuses.svg
Diagram showing major parts of a lymph node.
Blausen 0623 LymphaticSystem Female.png
Lymph nodes form part of the lymphatic system, and are present in most parts of the body, and connected by small lymphatic vessels.
Details
System Lymphatic system, part of the immune system
Identifiers
Latin nodus lymphaticus (singular); nodi lymphatici (plural)
MeSH D008198
TA98 A13.2.03.001
TA2 5192
FMA 5034
Anatomical terminology

A lymph node, or lymph gland, [1] is a kidney-shaped organ of the lymphatic system and the adaptive immune system. A large number of lymph nodes are linked throughout the body by the lymphatic vessels. They are major sites of lymphocytes that include B and T cells. Lymph nodes are important for the proper functioning of the immune system, acting as filters for foreign particles including cancer cells, but have no detoxification function.

Contents

In the lymphatic system a lymph node is a secondary lymphoid organ. A lymph node is enclosed in a fibrous capsule and is made up of an outer cortex and an inner medulla.

Lymph nodes become inflamed or enlarged in various diseases, which may range from trivial throat infections to life-threatening cancers. The condition of lymph nodes is very important in cancer staging, which decides the treatment to be used and determines the prognosis. Lymphadenopathy refers to glands that are enlarged or swollen. When inflamed or enlarged, lymph nodes can be firm or tender.

Structure

Cross-section of a lymph node with sections labelled.1) Capsule; 2) Subcapsular sinus; 3) Germinal centre; 4) Lymphoid nodule; 5) Trabeculae Lymphknoten (Schwein).jpg
Cross-section of a lymph node with sections labelled.1) Capsule; 2) Subcapsular sinus; 3) Germinal centre; 4) Lymphoid nodule; 5) Trabeculae

Lymph nodes are kidney or oval shaped and range in size from 2 mm to 25 mm on their long axis, with an average of 15 mm. [2]

Each lymph node is surrounded by a fibrous capsule, which extends inside a lymph node to form trabeculae. [3] The substance of a lymph node is divided into the outer cortex and the inner medulla. [3] These are rich with cells. [4] The hilum is an indent on the concave surface of the lymph node where lymphatic vessels leave and blood vessels enter and leave. [4]

Lymph enters the convex side of a lymph node through multiple afferent lymphatic vessels and from there flows into a series of sinuses. [3] After entering the lymph node from afferent lymphatic vessels, lymph flows into a space underneath the capsule called the subcapsular sinus, then into cortical sinuses. [3] After passing through the cortex, lymph then collects in medullary sinuses. [3] All of these sinuses drain into the efferent lymph vessels to exit the node at the hilum on the concave side. [3]

Location

Lymph nodes are present throughout the body, are more concentrated near and within the trunk, and are divided into groups. [4] There are about 450 lymph nodes in the adult. [4] Some lymph nodes can be felt when enlarged (and occasionally when not), such as the axillary lymph nodes under the arm, the cervical lymph nodes of the head and neck and the inguinal lymph nodes near the groin crease. Most lymph nodes lie within the trunk adjacent to other major structures in the body - such as the paraaortic lymph nodes and the tracheobronchial lymph nodes. The lymphatic drainage patterns are different from person to person and even asymmetrical on each side of the same body. [5] [6]

There are no lymph nodes in the central nervous system, which is separated from the body by the blood–brain barrier. Lymph from the meningeal lymphatic vessels in the CNS drains to the deep cervical lymph nodes. [7]

Size

Upper limit of lymph node sizes in adults
Generally10 mm [8] [9]
Inguinal10 [10] – 20 mm [11]
Pelvis 10 mm for ovoid lymph nodes, 8 mm for rounded [10]
Neck
Generally (non-retropharyngeal)10 mm [10] [12]
Jugulodigastric lymph nodes 11mm [10] or 15 mm [12]
Retropharyngeal8 mm [12]
  • Lateral retropharyngeal: 5 mm [10]
Mediastinum
Mediastinum, generally10 mm [10]
Superior mediastinum and high paratracheal7mm [13]
Low paratracheal and subcarinal11 mm [13]
Upper abdominal
Retrocrural space6 mm [14]
Paracardiac8 mm [14]
Gastrohepatic ligament8 mm [14]
Upper paraaortic region9 mm [14]
Portacaval space10 mm [14]
Porta hepatis7 mm [14]
Lower paraaortic region11 mm [14]

Subdivisions

Histology of a normal lymphoid follicle, showing dark, light, mantle and marginal zones. Dark, light, mantle and marginal zones of a secondary follicle.png
Histology of a normal lymphoid follicle, showing dark, light, mantle and marginal zones.

A lymph node is divided into compartments called nodules (or lobules), each consisting of a region of cortex with combined follicle B cells, a paracortex of T cells, and a part of the nodule in the medulla. [15] The substance of a lymph node is divided into the outer cortex and the inner medulla. [3] The cortex of a lymph node is the outer portion of the node, underneath the capsule and the subcapsular sinus. [15] It has an outer part and a deeper part known as the paracortex. [15] The outer cortex consists of groups of mainly inactivated B cells called follicles. [4] When activated, these may develop into what is called a germinal centre. [4] The deeper paracortex mainly consists of the T cells. [4] Here the T-cells mainly interact with dendritic cells, and the reticular network is dense. [16]

The medulla contains large blood vessels, sinuses and medullary cords that contain antibody-secreting plasma cells. There are fewer cells in the medulla. [4]

The medullary cords are cords of lymphatic tissue, and include plasma cells, macrophages, and B cells.

Cells

In the lymphatic system a lymph node is a secondary lymphoid organ. [4] Lymph nodes contain lymphocytes, a type of white blood cell, and are primarily made up of B cells and T cells. [4] B cells are mainly found in the outer cortex where they are clustered together as follicular B cells in lymphoid follicles, and T cells and dendritic cells are mainly found in the paracortex. [17]

There are fewer cells in the medulla than the cortex. [4] The medulla contains plasma cells, as well as macrophages which are present within the medullary sinuses. [17]

As part of the reticular network, there are follicular dendritic cells in the B cell follicle and fibroblastic reticular cells in the T cell cortex. The reticular network provides structural support and a surface for adhesion of the dendritic cells, macrophages and lymphocytes. It also allows exchange of material with blood through the high endothelial venules and provides the growth and regulatory factors necessary for activation and maturation of immune cells. [18]

Lymph flow

Labeled diagram of human lymph node showing the flow of lymph. Lymphatic immune system lymph node5-CROPPED.jpg
Labeled diagram of human lymph node showing the flow of lymph.
Afferent and efferent vessels Illu lymph node structure.png
Afferent and efferent vessels

Lymph enters the convex side of a lymph node through multiple afferent lymphatic vessels, which form a network of lymphatic vessels (Latin : plexus) and from here flows into a space (Latin : sinus) underneath the capsule called the subcapsular sinus. [4] [3] From here, lymph flows into sinuses within the cortex. [3] After passing through the cortex, lymph then collects in medullary sinuses. [3] All of these sinuses drain into the efferent lymphatic vessels to exit the node at the hilum on the concave side. [3]

These are channels within the node lined by endothelial cells along with fibroblastic reticular cells, allowing for the smooth flow of lymph. The endothelium of the subcapsular sinus is continuous with that of the afferent lymph vessel and also with that of the similar sinuses flanking the trabeculae and within the cortex. These vessels are smaller and do not allow the passage of macrophages so that they remain contained to function within a lymph node. In the course of the lymph, lymphocytes may be activated as part of the adaptive immune response.

There is usually only one efferent vessel though sometimes there may be two. [19] Medullary sinuses contain histiocytes (immobile macrophages) and reticular cells.

A lymph node contains lymphoid tissue, i.e., a meshwork or fibers called reticulum with white blood cells enmeshed in it. The regions where there are few cells within the meshwork are known as lymph sinus. It is lined by reticular cells, fibroblasts and fixed macrophages. [20]

Capsule

Lymph node tissue showing trabeculae Gray598.png
Lymph node tissue showing trabeculae

Thin reticular fibers (reticulin) of reticular connective tissue form a supporting meshwork inside the node. [4] The lymph node capsule is composed of dense irregular connective tissue with some plain collagenous fibers, and a number of membranous processes or trabeculae extend from its internal surface. The trabeculae pass inward, radiating toward the center of the node, for about one-third or one-fourth of the space between the circumference and the center of the node. In some animals they are sufficiently well-marked to divide the peripheral or cortical portion of the node into a number of compartments (nodules), but in humans this arrangement is not obvious. The larger trabeculae springing from the capsule break up into finer bands, and these interlace to form a mesh-work in the central or medullary portion of the node. These trabecular spaces formed by the interlacing trabeculae contain the proper lymph node substance or lymphoid tissue. The node pulp does not, however, completely fill the spaces, but leaves between its outer margin and the enclosing trabeculae a channel or space of uniform width throughout. This is termed the subcapsular sinus (lymph path or lymph sinus). Running across it are a number of finer trabeculae of reticular fibers, mostly covered by ramifying cells.

Function

In the lymphatic system a lymph node is a secondary lymphoid organ. [4]

Diagram of a lymph node showing lymphocytes. Diagram of a lymph node CRUK 022.svg
Diagram of a lymph node showing lymphocytes.

The primary function of lymph nodes is the filtering of lymph to identify and fight infection. In order to do this, lymph nodes contain lymphocytes, a type of white blood cell, which includes B cells and T cells. These circulate through the bloodstream and enter and reside in lymph nodes. [21] B cells produce antibodies. Each antibody has a single predetermined target, an antigen, that it can bind to. These circulate throughout the bloodstream and if they find this target, the antibodies bind to it and stimulate an immune response. Each B cell produces different antibodies, and this process is driven in lymph nodes. B cells enter the bloodstream as "naive" cells produced in bone marrow. After entering a lymph node, they then enter a lymphoid follicle, where they multiply and divide, each producing a different antibody. If a cell is stimulated, it will go on to produce more antibodies (a plasma cell) or act as a memory cell to help the body fight future infection. [22] If a cell is not stimulated, it will undergo apoptosis and die. [22]

Antigens are molecules found on bacterial cell walls, chemical substances secreted from bacteria, or sometimes even molecules present in body tissue itself. These are taken up by cells throughout the body called antigen-presenting cells, such as dendritic cells. [23] These antigen presenting cells enter the lymph system and then lymph nodes. They present the antigen to T cells and, if there is a T cell with the appropriate T cell receptor, it will be activated. [22]

B cells acquire antigen directly from the afferent lymph. If a B cell binds its cognate antigen it will be activated. Some B cells will immediately develop into antibody secreting plasma cells, and secrete IgM. Other B cells will internalize the antigen and present it to follicular helper T cells on the B and T cell zone interface. If a cognate FTh cell is found it will upregulate CD40L and promote somatic hypermutation and isotype class switching of the B cell, increasing its antigen binding affinity and changing its effector function. Proliferation of cells within a lymph node will make the node expand.

Lymph is present throughout the body, and circulates through lymphatic vessels. These drain into and from lymph nodes –afferent vessels drain into nodes, and efferent vessels from nodes. When lymph fluid enters a node, it drains into the node just beneath the capsule in a space called the subcapsular sinus. The subcapsular sinus drains into trabecular sinuses and finally into medullary sinuses. The sinus space is criss-crossed by the pseudopods of macrophages, which act to trap foreign particles and filter the lymph. The medullary sinuses converge at the hilum and lymph then leaves the lymph node via the efferent lymphatic vessel towards either a more central lymph node or ultimately for drainage into a central venous subclavian blood vessel.

Clinical significance

Swelling

A still image from a 3D medical animation showing enlarged lymph nodes. Swollen Lymph Nodes.jpg
A still image from a 3D medical animation showing enlarged lymph nodes.

Lymph node enlargement or swelling is known as lymphadenopathy. [24] Swelling may be due to many causes, including infections, tumors, autoimmune disease, drug reactions, diseases such as amyloidosis and sarcoidosis, or because of lymphoma or leukemia. [25] [24] Depending on the cause, swelling may be painful, particularly if the expansion is rapid and due to an infection or inflammation. [24] Lymph node enlargement may be localised to an area, which might suggest a local source of infection or a tumour in that area that has spread to the lymph node. [24] It may also be generalised, which might suggest infection, connective tissue or autoimmune disease, or a malignancy of blood cells such as a lymphoma or leukaemia. [24] Rarely, depending on location, lymph node enlargement may cause problems such as difficulty breathing, or compression of a blood vessel (for example, superior vena cava obstruction [26] ).

Enlarged lymph nodes might be felt as part of a medical examination, or found on medical imaging. [27] Features of the medical history may point to the cause, such as the speed of onset of swelling, pain, and other constitutional symptoms such as fevers or weight loss. [28] For example, a tumour of the breast may result in swelling of the lymph nodes under the arms [24] and weight loss and night sweats may suggest a malignancy such as lymphoma. [24]

In addition to a medical exam by a medical practitioner, medical tests may include blood tests and scans may be needed to further examine the cause. [24] A biopsy of a lymph node may also be needed. [24]

Cancer

Micrograph of a mesenteric lymph node with adenocarcinoma Crc met to node1.jpg
Micrograph of a mesenteric lymph node with adenocarcinoma

Lymph nodes can be affected by both primary cancers of lymph tissue, and secondary cancers affecting other parts of the body. Primary cancers of lymph tissue are called lymphomas and include Hodgkin lymphoma and non-Hodgkin lymphoma. [29] Cancer of lymph nodes can cause a wide range of symptoms from painless long-term slowly growing swelling to sudden, rapid enlargement over days or weeks, with symptoms depending on the grade of the tumour. [29] Most lymphomas are tumours of B-cells. [29] Lymphoma is managed by haematologists and oncologists.

Local cancer in many parts of the body can cause lymph nodes to enlarge because of tumorous cells that have metastasised into the node. [30] Lymph node involvement is often a key part in the diagnosis and treatment of cancer, acting as "sentinels" of local disease, incorporated into TNM staging and other cancer staging systems. As part of the investigations or workup for cancer, lymph nodes may be imaged or even surgically removed. If removed, the lymph node will be stained and examined under a microscope by a pathologist to determine if there is evidence of cells that appear cancerous (i.e. have metastasized into the node). The staging of the cancer, and therefore the treatment approach and prognosis, is predicated on the presence of node metastases.

Lymphedema

Lymphedema is the condition of swelling (edema) of tissue relating to insufficient clearance by the lymphatic system. [31] It can be congenital as a result usually of undeveloped or absent lymph nodes, and is known as primary lymphedema. Lymphedema most commonly arises in the arms or legs, but can also occur in the chest wall, genitals, neck, and abdomen. [32] Secondary lymphedema usually results from the removal of lymph nodes during breast cancer surgery or from other damaging treatments such as radiation. It can also be caused by some parasitic infections. Affected tissues are at a great risk of infection.[ citation needed ] Management of lymphedema may include advice to lose weight, exercise, keep the affected limb moist, and compress the affected area. [31] Sometimes surgical management is also considered. [31]

Similar lymphoid organs

The spleen and the tonsils are the larger secondary lymphoid organs that serve somewhat similar functions to lymph nodes, though the spleen filters blood cells rather than lymph. The tonsils are sometimes erroneously referred to as lymph nodes. Although the tonsils and lymph nodes do share certain characteristics, there are also many important differences between them, such as their location, structure and size. [33] Furthermore, the tonsils filter tissue fluid whereas lymph nodes filter lymph. [33]

The appendix contains lymphoid tissue and is therefore believed to play a role not only in the digestive system, but also in the immune system. [34]

See also

Related Research Articles

<span class="mw-page-title-main">Thymus</span> Endocrine gland

The thymus is a specialized primary lymphoid organ of the immune system. Within the thymus, thymus cell lymphocytes or T cells mature. T cells are critical to the adaptive immune system, where the body adapts to specific foreign invaders. The thymus is located in the upper front part of the chest, in the anterior superior mediastinum, behind the sternum, and in front of the heart. It is made up of two lobes, each consisting of a central medulla and an outer cortex, surrounded by a capsule.

<span class="mw-page-title-main">Lymphatic system</span> Organ system in vertebrates

The lymphatic system, or lymphoid system, is an organ system in vertebrates that is part of the immune system, and complementary to the circulatory system. It consists of a large network of lymphatic vessels, lymph nodes, lymphoid organs, lymphoid tissues and lymph. Lymph is a clear fluid carried by the lymphatic vessels back to the heart for re-circulation..

<span class="mw-page-title-main">Dendritic cell</span> Accessory cell of the mammalian immune system

A dendritic cell (DC) is an antigen-presenting cell of the mammalian immune system. A DC's main function is to process antigen material and present it on the cell surface to the T cells of the immune system. They act as messengers between the innate and adaptive immune systems.

<span class="mw-page-title-main">Lymphatic vessel</span> Tubular vessels that are involved in the transport of lymph and lymphocytes

The lymphatic vessels are thin-walled vessels (tubes), structured like blood vessels, that carry lymph. As part of the lymphatic system, lymph vessels are complementary to the cardiovascular system. Lymph vessels are lined by endothelial cells, and have a thin layer of smooth muscle, and adventitia that binds the lymph vessels to the surrounding tissue. Lymph vessels are devoted to the propulsion of the lymph from the lymph capillaries, which are mainly concerned with the absorption of interstitial fluid from the tissues. Lymph capillaries are slightly bigger than their counterpart capillaries of the vascular system. Lymph vessels that carry lymph to a lymph node are called afferent lymph vessels, and those that carry it from a lymph node are called efferent lymph vessels, from where the lymph may travel to another lymph node, may be returned to a vein, or may travel to a larger lymph duct. Lymph ducts drain the lymph into one of the subclavian veins and thus return it to general circulation.

Gut-associated lymphoid tissue (GALT) is a component of the mucosa-associated lymphoid tissue (MALT) which works in the immune system to protect the body from invasion in the gut.

<span class="mw-page-title-main">Marginal zone</span> Part of the spleen

The marginal zone is the region at the interface between the non-lymphoid red pulp and the lymphoid white-pulp of the spleen.

<span class="mw-page-title-main">Germinal center</span> Lymphatic tissue structure

Germinal centers or germinal centres (GCs) are transiently formed structures within B cell zone (follicles) in secondary lymphoid organs – lymph nodes, ileal Peyer's patches, and the spleen – where mature B cells are activated, proliferate, differentiate, and mutate their antibody genes during a normal immune response; most of the germinal center B cells (BGC) are removed by tingible body macrophages. There are several key differences between naive B cells and GC B cells, including level of proliferative activity, size, metabolic activity and energy production. The B cells develop dynamically after the activation of follicular B cells by T-dependent antigen. The initiation of germinal center formation involves the interaction between B and T cells in the interfollicular area of the lymph node, CD40-CD40L ligation, NF-kB signaling and expression of IRF4 and BCL6.

<span class="mw-page-title-main">Follicular dendritic cells</span> Immune cells found in lymph nodes

Follicular dendritic cells (FDC) are cells of the immune system found in primary and secondary lymph follicles of the B cell areas of the lymphoid tissue. Unlike dendritic cells (DC), FDCs are not derived from the bone-marrow hematopoietic stem cell, but are of mesenchymal origin. Possible functions of FDC include: organizing lymphoid tissue's cells and microarchitecture, capturing antigen to support B cell, promoting debris removal from germinal centers, and protecting against autoimmunity. Disease processes that FDC may contribute include primary FDC-tumor, chronic inflammatory conditions, HIV-1 infection development, and neuroinvasive scrapie.

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<span class="mw-page-title-main">CCL21</span> Mammalian protein found in Homo sapiens

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<span class="mw-page-title-main">CCL19</span> Mammalian protein found in Homo sapiens

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<span class="mw-page-title-main">C-C chemokine receptor type 7</span> Protein-coding gene in the species Homo sapiens

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<span class="mw-page-title-main">Mantle zone</span>

The mantle zone of a lymphatic nodule is an outer ring of small lymphocytes surrounding a germinal center.

<span class="mw-page-title-main">Marginal-zone B cell</span>

Marginal-zone B cells are noncirculating mature B cells that in humans segregate anatomically into the marginal zone (MZ) of the spleen and certain other types of lymphoid tissue. The MZ B cells within this region typically express low-affinity polyreactive B-cell receptors (BCR), high levels of IgM, Toll-like receptors (TLRs), CD21, CD1, CD9, CD27 with low to negligible levels of secreted-IgD, CD23, CD5, and CD11b that help to distinguish them phenotypically from follicular (FO) B cells and B1 B cells.

Lymph node stromal cells are essential to the structure and function of the lymph node whose functions include: creating an internal tissue scaffold for the support of hematopoietic cells; the release of small molecule chemical messengers that facilitate interactions between hematopoietic cells; the facilitation of the migration of hematopoietic cells; the presentation of antigens to immune cells at the initiation of the adaptive immune system; and the homeostasis of lymphocyte numbers. Stromal cells originate from multipotent mesenchymal stem cells.

<span class="mw-page-title-main">Follicular hyperplasia</span> Medical condition of lymphatic cells

Follicular hyperplasia (FH) is a type of lymphoid hyperplasia and is classified as a lymphadenopathy, which means a disease of the lymph nodes. It is caused by a stimulation of the B cell compartment and by abnormal cell growth of secondary follicles. This typically occurs in the cortex without disrupting the lymph node capsule. The follicles are pathologically polymorphous, are often contrasting and varying in size and shape. Follicular hyperplasia is distinguished from follicular lymphoma in its polyclonality and lack of bcl-2 protein expression, whereas follicular lymphoma is monoclonal, and expresses bcl-2.

<span class="mw-page-title-main">Lymphoid neoplasms with plasmablastic differentiation</span>

Lymphoid neoplasms with plasmablastic differentiation were classified by the World Health Organization, 2017 as a sub-grouping of several distinct but rare lymphomas in which the malignant cells are B-cell lymphocytes that have become plasmablasts, i.e. immature plasma cells. Normally, B-cells take up foreign antigens, move to the germinal centers of secondary lymphoid organs such the spleen and lymph nodes, and at these sites are stimulated by T-cell lymphocytes to differentiate into plasmablasts and thereafter mature plasma cells. Plasmablasts, and to a greater extent, plasma cells make and secrete antibodies that bind the antigens to which their predecessor B-cells were previously exposed. Antibodies function, in part, to neutralize harmful bacteria and viruses by binding antigens that are exposed on their surfaces. Due to their malignant nature, however, the plasmablasts in lymphoid neoplasms with plasmablastic differentiation do not mature into plasma cells or form antibodies but rather uncontrollably proliferate in and damage various tissues and organs. The individual lymphomas in this sub-group of malignancies have heterogeneous clinical, morphological, and gene findings that often overlap with other members of the sub-group. In consequence, correctly diagnosing these lymphomas has been challenging. Nonetheless, it is particularly important to diagnose them correctly because they often have very different prognoses and treatments. The lymphoid neoplasms with plasmacytic differentiation are:

<span class="mw-page-title-main">Gwendalyn J. Randolph</span> American immunologist

Gwendalyn J. Randolph is an American immunologist, the Emil R. Unanue Distinguished Professor in the Department of Immunology and Pathology at Washington University in St. Louis, Missouri, where she is currently co-director of the Immunology Graduate Program. During her postdoctoral work, Randolph characterized monocyte differentiation to dendritic cells and macrophages and made advances in our understanding of dendritic cell trafficking and the fate of monocytes recruited to sites of inflammation. Her lab has contributed to the Immunological Genome Project by characterizing macrophage gene expression. Her work now focuses on the immunological mechanisms driving atherosclerosis and inflammatory bowel disease (IBD) by exploring lymphatic function and lipoprotein trafficking.

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  26. ↑ Davidson's 2018, p. 1326.
  27. ↑ Gaddey, Heidi L.; Riegel, Angela M. (1 December 2016). "Unexplained Lymphadenopathy: Evaluation and Differential Diagnosis". American Family Physician. 94 (11): 896–903. ISSN   1532-0650. PMID   27929264.
  28. ↑ Davidson's 2018, p. 913.
  29. 1 2 3 Davidson's 2018, p. 961.
  30. ↑ Davidson's 2018, p. 1324.
  31. 1 2 3 Maclellan RA, Greene AK (August 2014). "Lymphedema". Seminars in Pediatric Surgery. 23 (4): 191–7. doi:10.1053/j.sempedsurg.2014.07.004. PMID   25241097.
  32. ↑ "Lymphedema". MayoClinic.org. 18 September 2019. Retrieved 17 November 2022.
  33. 1 2 Lakna (31 January 2019). "What is the Difference Between Tonsils and Lymph Nodes". Pediaa.Com. Retrieved 14 December 2019.
  34. ↑ Kooij IA, Sahami S, Meijer SL, Buskens CJ, Te Velde AA (October 2016). "The immunology of the vermiform appendix: a review of the literature". Clinical and Experimental Immunology. 186 (1): 1–9. doi:10.1111/cei.12821. PMC   5011360 . PMID   27271818.

Bibliography

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