To develop a nomogram to predict the prognosis of gastric cancer patients on the basis of metastatic lymph nodes ratio (mLNR), especially in the patients with total number of examined lymph nodes (TLN) less than 15. The nomogram was constructed based on a retrospective database that included 2,205 patients underwent curative resection in Cancer Center, Sun Yat-sen University (SYSUCC). Resectable gastric cancer (RGC) patients underwent curative resection before December 31, 2008 were assigned as the training set (n=1,470) and those between January 1, 2009 and December 31, 2012 were selected as the internal validation set (n=735). Additional external validations were also performed separately by an independent data set (n=602) from Jiangxi Provincial Cancer Hospital (JXCH) in Jiangxi, China and a data set (n=3,317) from the Surveillance, Epidemiology, and End Results (SEER) database. The Independent risk factors were identified by Multivariate Cox Regression. In the SYSUCC set, TNM (Tumor-node-metastasis) and TRM-based (Tumor-Positive Nodes Ratio-Metastasis) nomograms were constructed respectively. The TNM-based nomogram showed better discrimination than the AJCC-TNM staging system (C-index: 0.73 versus 0.69, p<0.01). When the mLNR was included in the nomogram, the C-index increased to 0.76. Furthermore, the C-index in the TRM-based nomogram was similar between TLN ≥16 (C-index: 0.77) and TLN ≤15 (C-index: 0.75). The discrimination was further ascertained by internal and external validations. We developed and validated a novel TRM-based nomogram that provided more accurate prediction of survival for gastric cancer patients who underwent curative resection, regardless of the number of examined lymph nodes.

Therapeutic delivery selectively to lymph nodes has the potential to address a variety of unmet clinical needs. However, owing to the unique structure of the lymphatics and the size-restrictive nature of the lymph node reticular network, delivering cargo to specific cells in the lymph node cortex and paracortex is difficult. Here, we describe a delivery system to overcome lymphatic and intra-lymph node transport barriers by combining nanoparticles that are rapidly conveyed to draining lymph nodes after administration in peripheral tissues with programmable degradable linkers. This platform enables the controlled release of intra-lymph-mobile small-molecular cargo, which can reach vastly more immune cells throughout the lymph node than either the particles or free compounds alone. The release rate can be programmed, allowing access to different lymph node structures and therefore specific lymphocyte subpopulations. We are thus able to alter the subtypes of drugged lymph node cells to improve immunotherapeutic effects.

The lymph nodal invasion diagnosis is critical for therapeutic-decision and follows up in gastric cancer. However, the number of nodes to be examined for nodal invasion diagnosis is still under controversy, and the model for quantifying risk of missing positive node is currently not reported yet. We analyzed the nodal invasion status of 13,857 gastric cancer samples with records of primary tumor stage, the number of examined and positive lymph nodes in the surveillance, epidemiology, and end results (SEER) database, fitting a beta-binomial model. The nodes need to be examined with different primary tumor stage were determined based on the model. Overall, examining 11 lymph nodes reduces the probability of missing positive nodes to <10%, and the currently median nodes dissected is adequate (12 nodes). While the number of nodes demands to be dissected for T1, T2, T3, and T4 subgroups are 6, 19, 40, and 66, respectively. The currently implemented median value for these samples was 12, 12, 13, and 16, separately. It implies that the number of nodes to be examined is sufficient for early gastric cancer (T1), but it is inadequate for middle and advanced gastric cancer (T2-T3). The clinical significance of nodal staging score was validated with survival information. In summary, we first quantified the lymph nodes to be examined during surgery using a beta-binomial model, and validated with survival information.

Females have more robust immune responses than males, well-illustrated by the degree of inflammation elicited during delayed-type hypersensitivity (DTH) responses. Here, we have investigated underlying sex differences that may contribute to differential footpad DTH responses using wildtype and four core genotypes (FCG) mice and popliteal lymphnode cellularity and gene expression. DTH responses in XX and XY FCG females showed no role for almost all genes expressed on sex chromosomes. After then filtering-out genes differentially expressed between XX and XY females, only one gene was sexually differentially expressed in wildtype mice, glycosylation-dependent cell adhesion molecule 1 (Glycam1), expressed 7-fold higher in females. Glycam1 facilitates leukocyte entry through high endothelial venules. Consistent with greater Glycam1 expression, female nodes contained twice as many cells. While females had more memory T cells in their nodes, males had a higher percentage of T regulatory cells. This sexual dimorphism in wildtype animals manifested pre-pubertally, was enhanced post-pubertally, and was eliminated by castration. The formation of male gonads is determined by the expression of Sry. Sry overexpression, which does not affect testosterone levels, produced an exaggerated male phenotype. We conclude that Sry expression through formation of the male gonad indirectly negatively impacts the potential for local inflammation.

Active-targeted delivery to lymph nodes represents a major advance toward more effective treatment of immune-mediated disease. The MECA79 antibody recognizes peripheral node addressin molecules expressed by high endothelial venules of lymph nodes. By mimicking lymphocyte trafficking to the lymph nodes, we have engineered MECA79-coated microparticles containing an immunosuppressive medication, tacrolimus. Following intravenous administration, MECA79-bearing particles showed marked accumulation in the draining lymph nodes of transplanted animals. Using an allograft heart transplant model, we show that targeted lymph node delivery of microparticles containing tacrolimus can prolong heart allograft survival with negligible changes in tacrolimus serum level. Using MECA79 conjugation, we have demonstrated targeted delivery of tacrolimus to the lymph nodes following systemic administration, with the capacity for immune modulation in vivo.

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 node mapping is important in cancer immunotherapy because the morphology of lymph nodes is one of the crucial evaluation criteria of immune responses. We developed new theragnostic glycol-chitosan-coated gold nanoparticles (GC-AuNPs), which highlighted lymph nodes in ultrasound-guided photoacoustic (US/PA) imaging. Moreover, the ovalbumin epitope was conjugated GC-AuNPs (OVA-GC-AuNPs) for delivering tumor antigen to lymph node resident macrophage. In vitro studies proved the vigorous endocytosis activity of J774A.1 macrophage and consequent strong photoacoustic signals from them. The macrophages also presented a tumor antigen when OVA-GC-AuNPs were used for cellular uptake. After the lingual injection of GC-AuNPs into healthy mice, cervical lymph nodes were visible in a US/PA imaging system with high contrast. Three-dimensional analysis of lymph nodes revealed that the accumulation of GC-AuNPs in the lymph node increased as the post-injection time passed. Histological analysis showed GC-AuNPs or OVA-GC-AuNPs located in subcapsular and medullar sinuses where macrophages are abundant. Our new theragnostic GC-AuNPs present a superior performance in US/PA imaging of lymph nodes without targeting moieties or complex surface modification. Simultaneously, GC-AuNPs were able to deliver tumor antigens to cause macrophages to present the OVA epitope at targeted lymph nodes, which would be valuable for cancer immunotherapy.

There are no established data about lymphadenectomy during treatment of endometrial cancers (ECs) and to what extent lymphadenectomy should be performed. In addition, retroperitoneal lymphadenectomy increases the intraoperative and postoperative complications. Sentinel lymph node (SLN) mapping has the lowest costs and highest quality-adjusted survival. SLN is the most cost-effective strategy in the management of low-risk ECs. Women staged with SLN mapping were more likely to receive adjuvant treatment compared with women staged with systemic lymphadenectomy. This review article designed to evaluate the diagnostic accuracy and the methods of SLN detection in ECs.

Background: Nodal status and tumor site are prognostic factors for resectable pancreatic ductal adenocarcinoma (PDAC). Parameters for nodal status are diverse, and the number of examined lymph nodes (eNs) needed for good prognosis are uncertain. We try to modify staging system of resectable PDAC with parameters mentioned above by recursive partitioning analysis. Methods: Patients from the Surveillance, Epidemiology, and End Results (SEER) database were divided into training cohort and internal validation cohort, randomly. PDAC patients from Sun Yat-sen University Cancer Center were regarded as external validation cohort. The training cohort was used to refine staging model by recursive partitioning analysis, while the internal validation cohort and the external validation cohort were applied to assess discriminatory capacity of staging model. For parameters included in the modified model, their effects were studied. Results: The number of eNs, tumor site and tumor size were risk factors for positive nodal status. Lymph nodes ratio (LNR), tumor site, eNs and T stages of 8th the American Joint Committee on Cancer (AJCC) were selected to develop a refined model, dividing patients into 5 groups of different outcomes, preceding 8th AJCC classification. Besides, we found that (1) for small PDAC (diameter < 1cm), lymph node metastasis was rarely found; (2) enough eNs were needed to ensure better prognosis of node-negative patients; (3) tumors in the head of pancreas were prone to lymph nodes metastasis; (4) for node-positive patients, LNR was a better nodal parameter compared to positive lymph nodes (pNs). Conclusion: Our improved staging system helps to illuminate the interactions among tumor site, size and eNs.

At present, only the number of metastatic lymph nodes (LNs+) is used for the pN category of AJCC TNM system for colon cancer. Recently, the ratio of metastatic to examined lymph nodes (LNR) has been reported to represent powerful independent predictive capacity in colon cancer. We sought to propose a novel category (nLN) which intergrades LNR and LNs+ into the AJCC staging system for colon cancer.

The value of adjuvant therapy in resected laryngeal cancer remains controversial. This large SEER-based cohort study aimed to investigate the existing parameters of lymph node status that could predict survival outcomes and the prognostic value of adjuvant treatment in resected laryngeal carcinoma.

Sheep are not only a major livestock species globally, they are also an important large animal model for biomedical research and have contributed to our understanding of the ontogeny and architecture of the mammalian immune system. In this study, we applied immunohistochemistry and multicolor immunofluorescence in fixed and paraffin-embedded lymph nodes to phenotype the key populations of antigen presenting cells, lymphocytes, and stromal cells that orchestrate the host adaptive immune response. We used an extensive panel of antibodies directed against markers associated with dendritic cells (MHC class II, CD83, and CD208), macrophages (CD11b, CD163, and CD169), stromal cells (CNA.42, S100, and CD83), and lymphocytes (CD3, Pax5, CD4, CD8). Using different methods of tissue fixation and antigen retrieval, we provide a detailed immunophenotyping of sheep lymph nodes including the identification of potential subpopulations of antigen presenting cells and stromal cells. By characterizing cells expressing combinations of these markers in the context of their morphology and location within the lymph node architecture, we provide valuable new tools to investigate the structure, activation, and regulation of the sheep immune system in health and disease.

The immune system defines a complex network of tissues and cell types that orchestrate responses across the body in a dynamic manner. The local and systemic interactions between immune and cancer cells contribute to disease progression. Lymphocytes are activated in lymph nodes, traffic through the periphery, and impact cancer progression through their interactions with tumor cells. As a result, therapeutic response and resistance are mediated across tissues, and a comprehensive understanding of lymphocyte dynamics requires a systems-level approach. In this review, we highlight experimental and computational methods that can leverage the study of leukocyte trafficking through an immunomics lens and reveal how adaptive immunity shapes cancer.

Observations that dendritic cells (DCs) constitutively enter afferent lymphatic vessels in many organs and that DCs in some tissues, such as the lung, turnover rapidly in the steady state have led to the concept that a major fraction of lymph node DCs are derived from migratory DCs that enter the lymph node through upstream afferent lymphatic vessels. We used the lysozyme M-Cre reporter mouse strain to assess the relationship of lymph node and nonlymphoid organ DCs. Our findings challenge the idea that a substantial proportion of lymph node DCs derive from the upstream tissue during homeostasis. Instead, our analysis suggests that nonlymphoid organ DCs comprise a major population of DCs within lymph nodes only after introduction of an inflammatory stimulus.

Elucidation of molecular pathways involved in development of human lymphoma requires efficient methods for tackling gene expression in lymph nodes. Expression studies of transcription factors in these malignancies facilitate understanding the changes occurring in neoplastic transformation and lymphoma development. Excised lymph nodes are routinely fixed in formalin and embedded in paraffin for diagnosis and stored in many hospitals' pathology archives. These tissues represent a precious resource for research since they allow retrospective studies to cover a broad range of human lymphoma even the less frequent types. Reverse transcription polymerase chain reaction (RT-PCR) is a commonly used method for gene expression analysis and a reproducible protocol for RNA isolation from lymph nodes is an inevitable requirement for these studies. However, formalin fixation and paraffin-embedding interfere with the quality of RNA especially when isolated from lymph nodes being the most fragile lymphatic tissues. We present here a simple and fast method for RNA isolation from formalin-fixed paraffin-embedded lymph nodes that can be successfully applied for RT-PCR as well as for quantitative RT-PCR analysis. We tested diverse isolation reagents and combined a range of factors in order to get a high quality RNA for retrospective studies of gene expression in human lymphoma samples. Our modified method of RNA extraction from FFPE provides superior yields and purity based on qPCR data.

To characterize the adhesion cascade that directs lymphocyte homing to peripheral lymph nodes (PLNs), we investigated the molecular mechanisms of lymphocyte interactions with the microvasculature of subiliac lymph nodes. We found that endogenous white blood cells and adoptively transferred lymph node lymphocytes (LNCs) tethered and rolled in postcapillary high endothelial venules (HEVs) and to a lesser extent in collecting venules. Similarly, firm arrest occurred nearly exclusively in the paracortical HEVs. Endogenous polymorphonuclear (PMNs) and mononuclear leukocytes (MNLs) attached and rolled in HEVs at similar frequencies, but only MNLs arrested suggesting that the events downstream of primary rolling interactions critically determine the specificity of lymphocyte recruitment. Antibody inhibition studies revealed that L-selectin was responsible for attachment and rolling of LNCs, and that LFA-1 was essential for sticking. LFA-1-dependent arrest was also abolished by pertussis toxin, implicating a requirement for G alpha i--protein-linked signaling. alpha 4 integrins, which play a critical role in lymphocyte homing to Peyer's Patches, made no significant contribution to attachment, rolling, or sticking in resting PLNs. Velocity analysis of interacting LNCs revealed no detectable contribution by LFA-1 to rolling. Taken together, our results suggest that lymphocyte- HEV interactions within PLNs are almost exclusively initiated by L-selectin followed by a G protein-coupled lymphocyte-specific activation event and activation-induced engagement of LFA-1. These events constitute a unique adhesion cascade that dictates the specificity of lymphocyte homing to PLNs.

The American College of Surgeons Oncology Group Z0011 trial reported that complete dissection of axillary lymph nodes (ALNs) may not be warranted in women with clinical T1-T2 tumors and one or two involved ALNs who were undergoing lumpectomy plus radiation followed by systemic therapy. The present study was conducted to identify preoperative imaging predictors of ≥ 3 ALNs.

To test the feasibility of localized intravaginal therapy directed to neighboring lymph nodes, the transport of quantum dots across the vaginal wall was investigated. Quantum dots instilled into the mouse vagina were transported across the vaginal mucosa into draining lymph nodes, but not into distant nodes. Most of the particles were transported to the lumbar nodes; far fewer were transported to the inguinal nodes. A low level of transport was evident at 4 hr after intravaginal instillation, and transport peaked at about 36 hr after instillation. Transport was greatly enhanced by prior vaginal instillation of Nonoxynol-9. Hundreds of micrograms of nanoparticles/kg tissue (ppb) were found in the lumbar lymph nodes at 36 hr post-instillation. Our results imply that targeted transport of microbicides or immunogens from the vagina to local lymph organs is feasible. They also offer an in vivo model for assessing the toxicity of compounds intended for intravaginal use.

Data evaluating the presence and characteristics of mesenteric lymph nodes (LNs) in patients with ulcerative colitis (UC) are scarce. The aim of this study is to determine the presence and characteristics of LNs in UC. The LN characteristics in computed tomography (CT), including LN dimension and attenuation, were evaluated retrospectively in 100 patients with UC (61 active and 39 inactive cases). Clinical characteristics and laboratory parameters, including CBC, biochemical analysis, erythrocyte sedimentation rate (ESR), and C reactive protein (CRP) were also compared. Mesenteric LNs were evident in all patients with UC. The attenuation and dimension of mesenteric LNs did not differ between active and inactive patients with UC. No correlation was found among patients with UC in terms of LN dimension, attenuation, ESR, CRP, leucocyte, and albumin (all with p > 0.05). The current study suggested that inflammation results in the development of mesenteric LN in UC, similar to Crohn’s disease and other inflammatory disorders.

This study aimed to characterize a soluble proteome of popliteal lymph nodes during lymphadenitis induced by intradermal injection of Bacillus anthracis Sterne spores in mice using tandem LC-MS/MS and reverse-phase protein microarray with antibodies specific to epitopes of phosphorylated proteins. More than 380 proteins were detected in the normal intra-nodal lymph, while the infectious process resulted in the profound changes in the protein abundances and appearance of 297 unique proteins. These proteins belong to an array of processes reflecting response to wounding, inflammation and perturbations of hemostasis, innate immune response, coagulation and fibrinolysis, regulation of body fluid levels and vascular disturbance among others. Comparison of lymph and serum revealed 83 common proteins. Also, using 71 antibodies specific to total and phosphorylated forms of proteins we carried initial characterization of circulating lymph phosphoproteome which brought additional information regarding signaling pathways operating in the lymphatics. The results demonstrate that the proteome of intra-nodal lymph serves as a sensitive sentinel of the processes occurring within the lymph nodes during infection. The acute innate response of the lymph nodes to anthrax is accompanied by cellular damage and inflammation with a large number of up- and down-regulated proteins many of which are distinct from those detected in serum. MS data are available via ProteomeXchange with identifier PXD001342.