The discovery of a new type of innervation in lymphoid organs.

It is well known that the main forms of innervation are synapses and free nerve endings, while other forms of innervation have not been reported. Here, we explore a new way of innervating lymphoid organs. Male Sprague-Dawley rats were used for studying the innervation of sympathetic nerve fibers in lymph nodes by means of anterograde tracking, immunoelectron microscopy, three-dimension reconstruction analysis, and immunofluorescence labeling. The results showed that the Fluoro-Ruby labeled nerve endings targeted only a group of cells in the lymph nodes and entered the cells through the plasma membrane. The electron microscopy showed that the biotinylated glucan amine reaction elements were distributed in the cytoplasm, and most of the biotinylated glucan amine active elements were concentrated on the microtubule and microfilament walls. Birbeck particles with rod-shaped and/or tennis racket like structures can be seen in the labeled cells at high magnification, and Birbeck particles contain biotinylated glucan amine-reactive elements. The immunofluoresence results showed that the Fluoro-Ruby-labeled nerve innervating cells expressed CD207 and CD1a protein. This result confirmed that the labeled cells were Langerhans cells. Our findings suggested that Langerhans cells might serve as a "bridge cell" for neuroimmune cross-talking in lymph organs, which play an important role in transmitting signals of the nervous system to immune system. This study also opened up a new way for further study of immune regulation mechanism.

Control of lymph node activity by direct local innervation.

The nervous system detects environmental and internal stimuli and relays this information to immune cells via neurotransmitters and neuropeptides. This is essential to respond appropriately to immunogenic threats and to support system homeostasis. Lymph nodes (LNs) act as sentinels where adaptive immune responses are generated. They are richly innervated by peripheral sympathetic and sensory nerves, which are responsible for the local secretion of neurotransmitters by sympathetic fibers, such as norepinephrine, and neuropeptides by sensory fibers, including calcitonin gene-related peptide (CGRP) and substance P. Additionally, time-of-day-dependent oscillations in nerve activity are associated with differential immune responses, suggesting a potential role for neuroimmune interactions in coordinating immunity in a circadian fashion. Here, we discuss how LN activity is controlled by local innervation.

Morphometric analysis of the human common hepatic artery reveals a rich and accessible target for sympathetic liver denervation.

This study quantified the distribution of nerves and adjacent anatomies surrounding human common hepatic artery (CHA) as guidance for catheter based denervation. CHA collected from cadaveric human donors (n = 20) were histologically evaluated and periarterial dimensions and distributions of nerves, lymph nodes, pancreas and blood vessels quantified by digital morphometry. Nerve abundance decreased significantly with distance from the aortic ostium (P < 0.0001) and was higher in the Superior/Inferior compared to the Anterior/Posterior quadrants (P = 0.014). In each locational group, nerves were absent from the artery wall, and starting 0.5-1.0 mm from the lumen exhibited a first order dependence on radial distance, fully defined by the median distance. Median subject-averaged nerve distance to the lumen was 2.75 mm, ranging from 2.1-3.1 mm in different arterial segments and quadrants and 2.0-3.5 mm in individuals. Inter-individual variance was high, with certain individuals exhibiting 50th and 75th nerve distances of, respectively, 3.5 and 6.5 mm The pancreas rarely approached within 4 mm of the lumen proximally and 2.5 mm more distally. The data indicate that the CHA is a rich and accessible target for sympathetic denervation regardless of sex and diabetes, with efficacy and safety most optimally balanced proximally.

Sympathetic nerve distribution in human lymph nodes.

Various lymph node functions are regulated by the sympathetic nervous system as shown in rodent studies. If human lymph nodes show a comparable neural regulation, their afferent nerves could represent a potential therapeutic target to treat, for example, infectious or autoimmune disease. Little information is available on human lymph node innervation and the aim of this study is to establish a comprehensive and accurate representation of the presence and location of sympathetic nerves in human lymph nodes. Since previous studies mention sympathetic paravascular nerves to occasionally extent into T cell-rich regions, the relation of these nerves with T cells was studied as well. A total number of 15 inguinal lymph nodes were resected from six donated human cadavers. Lymph node sections were stained with HE and a double T/B cell staining for evaluation of their morphology and to screen for general pathologies. A triple stain was used to identify blood vessels, sympathetic nerves and T cells, and, to study the presence and location of sympathetic nerves and their relation to T cells. To evaluate whether the observed nerves were en route to other structures or were involved in local processes, adjacent slides were stained with a marker for varicosities (synaptophysin), which presence is suggestive for synaptic activity. All lymph nodes contained sympathetic nerves, both as paravascular and discrete structures. In 15/15 lymph nodes, nerves were observed in their capsule, medulla and hilum, whereas only 13/15 lymph nodes contained nerves in their cortex. The amount of sympathetic nerves varied between compartments and between and within individuals. In general, if a lymph node contained more paravascular nerves in a specific compartment, more discrete nerves were observed as well. Occasionally, discrete nerves were observed in relation to T cells in lymphoid tissues of the cortex and medulla. Furthermore, discrete nerves were frequently present in the capsule and hilum. The presence of varicosities in a portion of these nerves, independently to their compartment, suggested a local regulatory function for these nerves. Human lymph nodes contain sympathetic nerves in their capsule, trabeculae, cortex, medulla and hilum, both as paravascular or as discrete structures. Discrete nerves were observed in relation to T cells and non-T cell-rich areas such as the hilar and capsular connective tissue. The presence of discrete structures suggests neural regulation of structures other than blood vessels, which was further supported by the presence of varicosities in a portion of these nerves. These observations are of relevance in further understanding neural regulation of lymph node immune responses and in the development of neuromodulatory immune therapies.

Lymph nodes are innervated by a unique population of sensory neurons with immunomodulatory potential.

Barrier tissue immune responses are regulated in part by nociceptors. Nociceptor ablation alters local immune responses at peripheral sites and within draining lymph nodes (LNs). The mechanisms and significance of nociceptor-dependent modulation of LN function are unknown. Using high-resolution imaging, viral tracing, single-cell transcriptomics, and optogenetics, we identified and functionally tested a sensory neuro-immune circuit that is responsive to lymph-borne inflammatory signals. Transcriptomics profiling revealed that multiple sensory neuron subsets, predominantly peptidergic nociceptors, innervate LNs, distinct from those innervating surrounding skin. To uncover LN-resident cells that may interact with LN-innervating sensory neurons, we generated a LN single-cell transcriptomics atlas and nominated nociceptor target populations and interaction modalities. Optogenetic stimulation of LN-innervating sensory fibers triggered rapid transcriptional changes in the predicted interacting cell types, particularly endothelium, stromal cells, and innate leukocytes. Thus, a unique population of sensory neurons monitors peripheral LNs and may locally regulate gene expression.

Adrenergic Innervation of the thyroid Gland, Blood and Lymph Vessels, and Lymph Nodes in Hypothyroidism.

Adrenergic innervation in the tissue of the thyroid gland, blood vessels of the thyroid gland, cervical lymphatic vessel, and lymph nodes in rats with hypothyroidism was studied by using a specific histochemical fluorescent-microscopic method of visualization of catecholamines. The presence of adrenergic innervation in the blood and lymph vessels and nodes was demonstrated. In hypothyroidism, diffusion of norepinephrine from nerve fibers and varicose thickenings was observed in the wall of the upper and lower thyroid arteries and adjacent cervical lymphatic vessels and nodes.

Immunofluorescence characterization of innervation and nerve-immune cell interactions in mouse lymph nodes.

The peripheral nervous system communicates specifically with the immune system via local interactions. These interactions include the "hardwiring" of sympathetic/parasympathetic (efferent) and sensory nerves (afferent) to primary (e.g., thymus and bone marrow) and secondary (e.g., lymph node, spleen, and gut-associated lymphoid tissue) lymphoid tissue/organs. To gain a better understanding of this bidirectional interaction/crosstalk between the two systems, we have investigated the distribution of nerve fibres and PNS-immune cell associations in situ in the mouse lymph node by using immunofluorescent staining and confocal microscopy/ three-dimensional reconstruction. Our results demonstrate i) the presence of extensive nerve fibres in all compartments (including B cell follicles) in the mouse lymph node; ii) close contacts/associations of nerve fibres with blood vessels (including high endothelial venules) and lymphatic vessels/sinuses; iii) close contacts/associations of nerve fibres with various subsets of dendritic cells (e.g., B220+CD11c+, CD4+CD11c+, CD8a+CD11c+, and Mac1+CD11c+), Mac1+ macrophages, and B/T lymphocytes. Our novel findings concerning the innervation and nerve-immune cell interactions inside the mouse lymph node should greatly facilitate our understanding of the effects that the peripheral nervous system has on cellular- and humoral-mediated immune responses or vice versa in health and disease.

Functional circuitry of neuro-immune communication in the mesenteric lymph node and spleen.

The peripheral nervous system is an active participant in immune responses capable of blocking aberrant activation of a variety of immune cells. As one of these neuro-immune circuits, the cholinergic anti-inflammatory pathway has been well established to reduce the severity of several immunopathologies. While the activation of this pathway by vagal nerve stimulation requires sympathetic innervation of the spleen, the neuro-immune circuitry remains highly controversial. Neuro-immune pathways in other lymphoid tissues such as mesenteric lymph nodes (MLN) that are critical to the surveillance of the small intestine and proximal colon have not been assessed. Using conditionally expressed Channelrhodopsin, selective stimulation of sympathetic post-ganglionic neurons in the superior mesenteric ganglion (SMG) prevented macrophage activation and LPS-induced TNFα production in the spleen and MLN, but not in the inguinal LN. Site selective stimulation of the SMG induced the release of norepinephrine, resulting in ß2AR dependent acetylcholine release in the MLN and spleen. VNS-evoked release of norepinephrine and acetylcholine in the MLN and spleen was significantly reduced using selective optogenetic blockade applied at the SMG. Additionally, this optogenetic blockade restored LPS-induced TNFα production, despite VNS. These studies identify the superior mesenteric ganglion as a critical node in a neuro-immune circuit that can inhibit immune function in the MLN and the spleen.

The role of perineural invasion in penile cancer: a meta-analysis and systematic review.

The significance of perineural invasion (PNI) present in penile cancer (PC) is controversial. In order to clarify the predictive role of PNI in the inguinal lymph node (ILN) metastases (ILNM) and oncologic outcome of patients, we performed this meta-analysis and systematic review. The search of PubMed, Embase, and Web of Science was conducted for appropriate studies, up to 20 January 2018. The pooled odds ratio (OR) and hazard ratio (HR) with their 95% confidence interval (CI) were applied to evaluate the difference in ILNM and oncologic outcome between patients present with PNI and those who were absent. A total of 298 in 1001 patients present with PNI were identified in current meta-analysis and systematic review. Significant difference was observed in ILNM between PNI present and absent from patients with PC (OR = 2.98, 95% CI = 2.00-4.45). Patients present with PNI had a worse cancer-specific survival (CSS) (HR = 3.58, 95% CI = 1.70-7.55) and a higher cancer-specific mortality (CSM) (HR = 2.20, 95% CI = 1.06-3.82) than those cases without PNI. This meta-analysis and systematic review demonstrated the predictive role of PNI in ILNM, CSS, and CSM for PC patients.

The Generation and Use of Allergen-Specific TCR Transgenic Animals.

The generation of allergen-specific TCR transgenic animals allows for the characterization of allergen-specific T-cell responses in vivo and in vitro and is a powerful tool to study adaptive immunity to allergens. Here we describe an approach starting from the isolation of antigen-specific T-cell hybridomas and using PCR, flow cytometric, and co-culture methods to obtain antigen-specific MHC class II-restricted CD4+ TCR transgenic mice on the Rag2-/- background.

Quantity of Lymph Nodes in the Vascularized Lymph Node Transfer Influences Its Lymphaticovenous Drainage.

BACKGROUND: The purpose of this study was to: (1) evaluate the mechanism of lymph drainage through a vascularized lymph node (VLN) flap, and (2) investigate if the number of VLNs impacts lymph transit time through the flap. METHODS: Twenty-seven axillary VLN flaps were elevated in 14 Sprague-Dawley rats and divided into three groups (n = 9 each) based on the number of lymph nodes present: group 1 (0-VLNs), group 2 (2-VLNs), and group 3 (4-VLNs). Indocyanine green (n = 8/group) and Alexa680-albumin (n = 1/group) were injected into the edge of flaps and the latency period between injection and fluorescence in the axillary vein was recorded. Stereomicroscopic fluorescent lymphography was performed to directly visualize lymphatic transit through VLNs. RESULTS: Fluorescence was detected in the axillary vein after 229s [47-476], 79s [15-289], and 56s [16-110] in group 1, 2, and 3, respectively (p < 0.01). There was a negative correlation between the number of VLNs in the flap and the latency period (r = -0.59; p < 0.05). Median flap weights were comparable in group 1, 2, and 3 (258 mg [196-349], 294 mg [212-407], 315 mg [204-386], respectively; p = 0.54). Stereoscopic lymphography allowed direct visualization of lymphatic fluid transit through VLNs. CONCLUSION: Lymphatic fluid in VLN flaps drains into the venous system mainly by passing through the afferent lymphatics and lymph nodes. A secondary mechanism appears to be the diffusion of fluid into the venous system via intratissue lymphaticovenous connections created during flap elevation. Increasing the number of lymph nodes in the flap is associated with a more rapid transit of fluid.

Intralymphatic nevus cells in benign nevi.

The histogenesis of nevus cell aggregates in lymph nodes lesion is controversial, and various hypotheses have been used to explain their origin. One of them is the transport of cells from cutaneous nevi or lesions to lymph nodes, called mechanical transport theory. We investigated in our cases of benign nevi to obtain evidence to substantiate this theory. A total of 369 benign cutaneous nevi were prospectively evaluated in excisional biopsy samples. Immunohistochemical stainings for CD31 and podoplanin (D2-40) were performed in the cases with intralymphatic nevus cell aggregate (ILNA), suspected for ILNA, and/or intralymphatic nevus cell protrusion. A total of 13 ILNAs were found in 10 patients. Six ILNA were verified with their histology as well as immunohistochemically with D2-40 and CD31. Protrusions of nevus cells inside the lymphatics (intralymphatic nevus cell protrusion) were seen in all cases of ILNA and also in 27 nevi where an ILNA was not observed. In most nevi, the perilymphatic orientation of nevus cells and their affinity to the lymphatics were observed. We suggested that ILNAs can be dislodged with local minor trauma and be carried inside the lymphatic vessel to the draining lymph node. Besides, whether ILNA or not, nevus cells could also move toward lymphatic spaces with mechanical effects due to their affinity to lymphatics and their localizations that are very close to the endothelium. Our findings might support the mechanical transport theory.

Social instability and immunity in rhesus monkeys: the role of the sympathetic nervous system.

Social instability can adversely affect endocrine, immune and health outcomes, and recent evidence suggests that the sympathetic nervous system (SNS) might mediate these effects. We conducted two studies with adult male rhesus monkeys (Macaca mulatta) to understand how social conditions affect measures of SNS activity and immune function. In Experiment 1, animals were socialized in stable social conditions, then were switched to unstable (stressful) social conditions, then were returned to stable conditions. Analysis revealed quadratic effects for measures of behaviour, urinary metabolites of epinephrine and norepinephrine, and expression of immune response genes: as expected, social instability adversely impacted most measures, and the effects remediated upon re-imposition of stable conditions. Cortisol levels were unaffected. In Experiment 2, we used the sympathomimetic drug methamphetamine to challenge the SNS; animals also underwent socialization in stable or unstable groups. Surprisingly, while methamphetamine elevated plasma catecholamines, responses in lymph nodes tracked the social, and not the drug, condition: social instability upregulated the density of SNS fibres in lymph nodes and downregulated Type I interferon gene expression. Together, these results indicate that the SNS is extremely sensitive to social conditions; full understanding of the adverse effects of social instability on health should therefore incorporate measures of this health-relevant system.

From theory to evidence: long-term evaluation of the mechanism of action and flap integration of distal vascularized lymph node transfers.

BACKGROUND: Nonanatomic (distal) placement of vascularized lymph node (VLN) transfers have shown efficacy in the treatment of extremity lymphedema, but the mechanism by which these flaps provide relief of lymphedema remains unclear. Intrinsic lymphovenous connections have been previously shown to exist in the transferred flap. But, the long-term interaction of the VLN flap and surrounding lymphedematous extremity has not been previously investigated. PATIENTS AND METHODS: A retrospective review of a prospective maintained database of patients who underwent VLN transfer was evaluated. Patients who underwent distal VLN transfer and had more than 1-year follow-up were identified. Lymphodynamic evaluation was performed using 0.3 to 0.6 mL indocyanine green (ICG) injection at 5 cm proximal to the flap edge on identified patients. Migration direction of dye and latency period was evaluated. RESULTS: In total, 20 patients were identified who met inclusion criteria. Average long-term follow-up was 27.3 months. The average circumference reduction of the affected extremity was 40.5%. ICG appearance within the VLN flap was found in all patients occurring on average in 178.3 seconds. In all cases, flow occurred in the distal direction (toward the flap) with proximal placement of dye. Latency period was found to inversely correlate with circumference reduction (p < 0.01). CONCLUSIONS: Distal, nonanatomic placement of VLN flaps provide sustained limb circumference reduction in extremity lymphedema patients following a minimum of 1-year postoperatively. Flap integration with the recipient site reliably occurs as witnessed with consistent ICG drainage, and occurs in the gravity-dependent direction. Faster clearance of ICG will result in improved clinical limb circumference reduction.

A Prospective Evaluation of Lymphedema-Specific Quality-of-Life Outcomes Following Vascularized Lymph Node Transfer.

BACKGROUND: Microsurgical techniques for the treatment of lymphedema rapidly increased in popularity. Although surgical success with vascularized lymph node (VLN) transfer has been demonstrated, limited studies have investigated the influence of microsurgical treatments on health-related quality-of-life (HRQoL) parameters. The aim of this study was to prospectively evaluate changes in HRQoL following VLN transfer for upper- and lower-extremity lymphedema using a validated instrument. METHODS: An Institutional Review Board-approved prospective study was performed of patients who underwent VLN transfer for symptomatic upper- or lower-limb lymphedema. A validated lymphedema-specific questionnaire-lymphoedema quality-of-life study-was utilized to assess specific quality-of-life parameters at multiple time points during the 12-month perioperative period. For a comparison with HRQoL metrics, limb circumference measurements were obtained to assess circumference differentiation. RESULTS: Twenty-five patients met the study criteria. Limb circumference analysis revealed significant early improvements following VLN transfer, with continued improvement during the study period (upper-limb lymphedema: 24.4 %; lower-limb lymphedema: 35.2 %). These improvements were mirrored by improvements in all HRQoL domains and overall quality of life (p < 0.01). The function, body appearance, symptom, and mood domains were all found to be significantly improved during the postoperative evaluation, with continued improvement being reported throughout the study period (p < 0.01 within each domain). CONCLUSIONS: Microsurgical treatment of lymphedema with VLN transfer procedures effectively decrease limb circumference. This improvement is mirrored by improvements in patient-reported outcomes and quality of life. These changes can be observed as soon as 1 month postoperatively, and continued steady improvement can be expected.

S100+ cells: a new neuro-immune cross-talkers in lymph organs.

Up to now, the 'hardwired' neural pathway of the neuro-immune regulation is not fully understood. Here we reported a new neural pathway which links sympathetic nerves with immune cells of the lymphoid tissues. Our results demonstrated that nerve fibers derived from superior cervical ganglion directly targeted only S100(+) cells in the cervical lymph nodes. Moreover, we found co-expression of neurotransmitters such as norepinephrine, vasoactive intestinal polypeptide and neuropeptide Y in the postganglionic sympathetic nerve endings that innervate S100(+) cells. Our findings suggested that S100(+) cells serve as a neuro-immune cross-talker in lymph organs that may play a significant role in transmitting signals of nervous cells to targeted immune cells. The new findings provide better understanding of the cross-talk mechanism between the nervous system and the immune system.

Monocytoid B cells: an enigmatic B cell subset showing evidence of extrafollicular immunoglobulin gene somatic hypermutation.

Monocytoid B cells are IgM(+) , IgD(-/+) , CD27(-) B cells, localized in the perisinusoidal area of the lymph node. These cells are especially prominent in infections such as those caused by toxoplasma and HIV. The ontogeny of monocytoid B cells with respect to B cell maturation is incompletely known. We analysed clonal expansion, somatic hypermutation and expression of activation-induced cytidine deaminase (AID) in monocytoid B cells. Sequence analysis of the rearranged immunoglobulin heavy chain genes amplified from microdissected monocytoid B cell zones with a high proportion of proliferating cells reveals the presence of multiple clones with low-level ongoing mutations (mean frequency: 0.46 × 10(-2) per bp). Mutation analysis of these ongoing mutations reveals strand bias, a preference of transitions over transversions as well as the occurrence of small deletions, as observed for somatically mutated immunoglobulin genes in the human germinal centre. Proliferation, ongoing mutation as well as expression of AID, combined, is evidence that monocytoid B cells acquire the mutations in the extrafollicular perisinusoidal area of the lymph node and pleads against a postgerminal centre B cell origin.

Increased density of sympathetic nerve fibers in metabolically activated fat tissue surrounding human synovium and mouse lymph nodes in arthritis.

OBJECTIVE: To investigate the density of sympathetic nerve fibers in and the metabolic activation of fat tissue surrounding human synovium in rheumatoid arthritis (RA)/osteoarthritis (OA) and in the draining lymph nodes of arthritic and normal mice. METHODS: Using immunofluorescence and immunohistochemistry, the density of sympathetic nerve fibers and the presence of nerve repellent factors were investigated. The metabolic activation of fat tissue was estimated by the occurrence of small-vacuole adipocytes, expression of ß3-adrenoceptors, and adipose tissue weight. RESULTS: The density of sympathetic nerve fibers was markedly increased in fat tissue surrounding RA synovium compared with that in fat tissue surrounding OA synovium. In adipose tissue adjacent to draining lymph nodes, the density of sympathetic nerve fibers was higher in arthritic mice compared with normal mice. In human synovium and mouse draining lymph nodes, the 2 sympathetic nerve repellent factors, semaphorin 3C and semaphorin 3F, were highly expressed. In arthritic compared with normal mice, the fat tissue around lymph nodes was markedly lighter, adipocytes had more fragmented lipid droplets, and fat tissue demonstrated high expression of ß3-adrenoceptors. CONCLUSION: This study demonstrated an increased density of sympathetic nerve fibers in metabolically activated fat tissue surrounding human RA synovium and the draining lymph nodes of arthritic mice. Because sympathetic neurotransmitters stimulate lipolysis, the repulsion of sympathetic nerve fibers from inflamed regions and their increased occurrence in fat tissue probably represent an adaptive program to support the proinflammatory process by releasing energy-rich substrates.

[Effects of stimulation of nervous fibres upon contractile activity of the tracheobronchial lymphatic node capsules' smooth muscles].

The contractile activity of smooth muscle of tracheobronchial lymph nodes' capsules was recorded in vitro. The field electric stimulation (0.5 ms pulses, 55 V nominal, 4 min trains at frequencies 0.5, 1, 2, 4, 8, 16 and 32 Hz) of strips from lymph node produced a frequency-dependent increase in baseline tension and frequency of phase contractions. Evoked contractions were significantly (about 80%) reduced by tetrodotoxin (1 x 10(-6) M). The blockage of M-cholinoreceptors with atropine (1 x 10(-6) M) did not affect the field-evoked responses. Contractile field-evoked effects were significantly reduced by the phentolamine (1 x 10(-7)-1 x 10(-6) M) but not completely. Field-evoked contractions were slightly affected by the propranolol (1 x 10(-7)-1 x 10(-6) M). We conclude that the contractile activity of bovine tracheobronchial lymph node capsular smooth muscle is modulated by excitatory adrenergic nerves.