Methylene blue and carbon nanoparticles had no significant adverse effects on the survival and function of lymph nodes after non-vascularized transplantation in a mouse model.

BACKGROUND: Lymph node transfer has been known as a physiologic approach for "curing" lymphedema. The transplantation of methylene blue and carbon nanoparticles stained lymph nodes aims to investigate their impact on the survival of transplanted lymph nodes. METHODS: Four weeks after transplantation, the survival condition of transplanted lymph nodes was detected by the reconnection with lymphatics, reconnected lymphatic function, HE staining, and distribution of B-cells and T-cells. Also, the number of lymphatics (LYVE-1) and blood vessels (CD31) of transplanted lymph nodes was investigated. RESULTS: Reconnected lymphatic function, the number of transplanted lymph nodes of achieving lymphatic reconstruction, and the surviving and potentially surviving ones were close. Besides, the control group (n = 11, CD31: mean = 9.527 ± 1.017, LYVE-1: mean = 21.45 ± 1.780), the methylene blue group (n = 12, CD31: mean = 9.73 ± 0.8998, LYVE-1: mean = 20.67 ± 1.601) and the carbon nanoparticles group (n = 11, CD31:mean = 8.709 ± 1.435, LYVE-1: mean = 19.60 ± 2.268). After calculation, both CD31 and LYVE-1 showed no significant difference statistically among the three groups. CONCLUSIONS: Our findings justify the use of methylene blue and carbon nanoparticles in reverse lymphatic mapping, as they can increase the efficiency of collecting "target lymph nodes" and minimize the iatrogenic injury in the donor area while having no significant adverse effects on the survival condition of transplanted lymph nodes. And this approach is easy to operate and worthy of clinical application and popularization.

Prognostic impact of lymph node micrometastasis in patients with gastric cancer.

PURPOSE: The clinical significance of lymph node micrometastasis (LNMM) remains controversial in gastric cancer (GC). In this study, we investigated the prognostic impact of LNMM in patients with GC. METHODS: A total of 624 patients with pathologically lymph node metastasis-negative (pN0) and N1 status (pN1) who underwent gastrectomy between 2004 and 2018 were enrolled in this retrospective study. The diameter of tumor cell clusters in metastatic lymph nodes was measured in 120 patients with pN1 GC. RESULTS: Patients with lymph node tumors < 1500 µm in diameter (LNMM) had a significantly better prognosis than those with tumors ≥ 1500 µm in diameter (p = 0.012; log-rank test). Cox's proportional hazards model revealed that LNMM (p = 0.016), several dissected lymph nodes (p = 0.049), and the provision of adjuvant chemotherapy (p = 0.002) were independent prognostic factors for the overall survival of patients with pN1 GC. There was no significant difference in the overall survival between patients with LNMM who received chemotherapy and those who did not (p = 0.332). CONCLUSIONS: LNMM is associated with a favorable prognosis and maybe an independent prognostic marker in patients with pN1 GC. LNMM in GC may be considered a factor preventing adjuvant chemotherapy.

Lymph node formation and B cell homeostasis require IKK-α in distinct endothelial cell-derived compartments.

Global inactivation of IκB kinase (IKK)-α results in defective lymph node (LN) formation and B cell maturation, and loss of IKK-α-dependent noncanonical NF-κB signaling in stromal organizer and hematopoietic cells is thought to underlie these distinct defects. We previously demonstrated that this pathway is also activated in vascular endothelial cells (ECs). To determine the physiologic function of EC-intrinsic IKK-α, we crossed IkkαF/F mice with Tie2-cre or Cdh5-cre mice to ablate IKK-α in ECs. Notably, the compound defects of global IKK-α inactivation were recapitulated in IkkαTie2 and IkkαCdh5 mice, as both lacked all LNs and mature follicular and marginal zone B cell numbers were markedly reduced. However, as Tie2-cre and Cdh5-cre are expressed in all ECs, including blood forming hemogenic ECs, IKK-α was also absent in hematopoietic cells (HC). To determine if loss of HC-intrinsic IKK-α affected LN development, we generated IkkαVav mice lacking IKK-α in only the hematopoietic compartment. While mature B cell numbers were significantly reduced in IkkαVav mice, LN formation was intact. As lymphatic vessels also arise during development from blood ECs, we generated IkkαLyve1 mice lacking IKK-α in lymphatic ECs (LECs) to determine if IKK-α in lymphatic vessels impacts LN development. Strikingly, while mature B cell numbers were normal, LNs were completely absent in IkkαLyve1 mice. Thus, our findings reveal that IKK-α in distinct EC-derived compartments is uniquely required to promote B cell homeostasis and LN development, and we establish that LEC-intrinsic IKK-α is absolutely essential for LN formation.

Development of follicular dendritic cells in lymph nodes depends on retinoic acid-mediated signaling.

Specialized stromal cells occupy and help define B- and T-cell domains, which are crucial for proper functioning of our immune system. Signaling through lymphotoxin and TNF receptors is crucial for the development of different stromal subsets, which are thought to arise from a common precursor. However, mechanisms that control the selective generation of the different stromal phenotypes are not known. Using in vitro cultures of embryonic mouse stromal cells, we show that retinoic acid-mediated signaling is important for the differentiation of precursors towards the Cxcl13pos follicular dendritic cell (FDC) lineage, and also blocks lymphotoxin-mediated Ccl19pos fibroblastic reticular cell lineage differentiation. Accordingly, at the day of birth we observe the presence of Cxcl13posCcl19neg/low and Cxcl13neg/lowCcl19pos cells within neonatal lymph nodes. Furthermore, ablation of retinoic acid receptor signaling in stromal precursors early after birth reduces Cxcl13 expression, and complete blockade of retinoic acid signaling prevents the formation of FDC networks in lymph nodes.

Vaccine delivery systems toward lymph nodes.

Strategies of improving vaccine targeting ability toward lymph nodes have been attracting considerable interest in recent years, though there are remaining delivery barriers based on the inherent properties of lymphatic systems and limited administration routes of vaccination. Recently, emerging vaccine delivery systems using various materials as carriers are widely developed to achieve efficient lymph node targeting and improve vaccine-triggered adaptive immune response. In this review, to further optimize the vaccine targeting ability for future research, the design principles of lymph node targeting vaccine delivery based on the anatomy of lymph nodes and vaccine administration routes are first summarized. Then different designs of lymph node targeting vaccine delivery systems, including vaccine delivery systems in clinical applications, are carefully surveyed. Also, the challenges and opportunities of current delivery systems for vaccines are concluded in the end.

Presence of Donor Lymph Nodes Within Vascularized Composite Allotransplantation Ameliorates VEGF-C-mediated Lymphangiogenesis and Delays the Onset of Acute Rejection.

BACKGROUND: The lymphatic system plays an active role in modulating inflammation in autoimmune diseases and organ rejection. In this work, we hypothesized that the transfer of donor lymph node (LN) might be used to promote lymphangiogenesis and influence rejection in vascularized composite allotransplantation (VCA). METHODS: Hindlimb transplantations were performed in which (1) recipient rats received VCA containing donor LN (D:LN+), (2) recipient rats received VCA depleted of all donor LN (D:LN-), and (3) D:LN+ transplantations were followed by lymphangiogenesis inhibition using a vascular endothelial growth factor receptor-3 (VEGFR3) blocker. RESULTS: Our data show that graft rejection started significantly later in D:LN+ transplanted rats as compared to the D:LN- group. Moreover, we observed a higher level of VEGF-C and a quicker and more efficient lymphangiogenesis in the D:LN+ group as compared to the D:LN- group. The presence of donor LN within the graft was associated with reduced immunoactivation in the draining LN and increased frequency of circulating and skin-resident donor T regulatory cells. Blocking of the VEGF-C pathway using a VEGFR3 blocker disrupts the lymphangiogenesis process, accelerates rejection onset, and interferes with donor T-cell migration. CONCLUSIONS: This study demonstrates that VCA LNs play a pivotal role in the regulation of graft rejection and underlines the potential of specifically targeting the LN component of a VCA to control graft rejection.

Comparing survival outcomes for cervical cancer based on the 2014 and 2018 International Federation of Gynecology and Obstetrics staging systems.

The International Federation of Gynecology and Obstetrics (FIGO) cervical cancer staging system was modified in 2018, introducing new stage IB subdivisions and new lymph node status considerations in stage IIIC. We compared cervical cancer survival outcomes according to the 2014 and 2018 FIGO staging systems. We selected 10% of cervical cancer cases (2010-2015) from the Korean national cancer registry (2010-2015) through a systematic sampling method. We collected information using a collaborative stage data collection system and evaluated the results according to both staging systems. The log-rank test was used to analyze overall survival differences. No significant difference in survival was observed between 2018 subdivisions IB1/IB2/IB3 (P = 0.069), whereas a considerable difference was observed between these subdivisions according to histological subtypes. In the 2018 FIGO staging system, stage IIIC had better survival than stage IIIA/IIIB (P < 0.001). We observed considerable heterogeneity in 2018 stage IIIC related to the corresponding stages of the 2014 staging system (stages IA1-IIIB). The size of the primary cervical mass was related to survival (P < 0.001). In conclusion, using lymph node status to define stage IIIC captured a broad range of prognoses. The inclusion of primary tumor size considerations may improve the staging accuracy of advanced cervical cancer.

The Lymph Node Reservoir: Physiology, HIV Infection, and Antiretroviral Therapy.

Despite advances in treatment, finding a cure for HIV remains a top priority. Chronic HIV infection is associated with increased risk of comorbidities, such as diabetes and cardiovascular disease. Additionally, people living with HIV must remain adherent to daily antiretroviral therapy, because lapses in medication adherence can lead to viral rebound and disease progression. Viral recrudescence occurs from cellular reservoirs in lymphoid tissues. In particular, lymph nodes are central to the pathology of HIV due to their unique architecture and compartmentalization of immune cells. Understanding how antiretrovirals (ARVs) penetrate lymph nodes may explain why these tissues are maintained as HIV reservoirs, and how they contribute to viral rebound upon treatment interruption. In this report, we review (i) the physiology of the lymph nodes and their function as part of the immune and lymphatic systems, (ii) the pathogenesis and outcomes of HIV infection in lymph nodes, and (iii) ARV concentrations and distribution in lymph nodes, and the relationship between ARVs and HIV in this important reservoir.

Differential activation of JAK-STAT signaling reveals functional compartmentalization in Drosophila blood progenitors.

Blood cells arise from diverse pools of stem and progenitor cells. Understanding progenitor heterogeneity is a major challenge. The Drosophila larval lymph gland is a well-studied model to understand blood progenitor maintenance and recapitulates several aspects of vertebrate hematopoiesis. However in-depth analysis has focused on the anterior lobe progenitors (AP), ignoring the posterior progenitors (PP) from the posterior lobes. Using in situ expression mapping and developmental and transcriptome analysis, we reveal PP heterogeneity and identify molecular-genetic tools to study this abundant progenitor population. Functional analysis shows that PP resist differentiation upon immune challenge, in a JAK-STAT-dependent manner. Upon wasp parasitism, AP downregulate JAK-STAT signaling and form lamellocytes. In contrast, we show that PP activate STAT92E and remain undifferentiated, promoting survival. Stat92E knockdown or genetically reducing JAK-STAT signaling permits PP lamellocyte differentiation. We discuss how heterogeneity and compartmentalization allow functional segregation in response to systemic cues and could be widely applicable.

Monocytic myeloid-derived suppressor cells home to tumor-draining lymph nodes via CCR2 and locally modulate the immune response.

Efficient priming of anti-tumor T cells requires the uptake and presentation of tumor antigens by immunogenic dendritic cells (DCs) and occurs mainly in lymph nodes draining the tumor (tdLNs). However, tumors expand and activate myeloid-derived suppressor cells (MDSCs) that inhibit CTL functions by several mechanisms. While the immune-suppressive nature of the tumor microenvironment is largely documented, it is not known whether similar immune-suppressive mechanisms operate in the tdLNs. In this study, we analyzed MDSC characteristics within tdLNs. We show that, in a metastasis-free context, MO-MDSCs are the dominant MDSC population within tdLNs, that they are highly suppressive and that tumor proximity enhances their recruitment to tdLN via a CCR2/CCL2-dependent pathway. Altogether our results uncover a mechanism by which tumors evade the immune system that involves MDSC-mediated recruitment to the tdLN and the inhibition of T-cell activation even before reaching the highly immunosuppressive tumor microenvironment.

Lymphatic immunomodulation using engineered drug delivery systems for cancer immunotherapy.

Though immunotherapy has revolutionized the treatment of cancer to improve disease outcomes, an array of challenges remain that limit wider clinical success, including low rate of response and immune-related adverse events. Targeting immunomodulatory drugs to therapeutically relevant tissues offers a way to overcome these challenges by potentially enabling enhanced therapeutic efficacy and decreased incidence of side effects. Research highlighting the importance of lymphatic tissues in the response to immunotherapy has increased interest in the application of engineered drug delivery systems (DDSs) to enable specific targeting of immunomodulators to lymphatic tissues and cells that they house. To this end, a variety of DDS platforms have been developed that enable more efficient uptake into lymphatic vessels and lymph nodes to provide targeted modulation of the immune response to cancer. This can occur either by delivery of immunotherapeutics to lymphatics tissues or by direct modulation of the lymphatic vasculature itself due to their direct involvement in tumor immune processes. This review will highlight DDS platforms that, by enabling the activities of cancer vaccines, chemotherapeutics, immune checkpoint blockade (ICB) antibodies, and anti- or pro-lymphangiogenic factors to lymphatic tissues through directed delivery and controlled release, augment cancer immunotherapy.

Regulation of lymphatic function and injury by nitrosative stress in obese mice.

OBJECTIVE: Obesity results in lymphatic dysfunction, but the cellular mechanisms that mediate this effect remain largely unknown. Previous studies in obese mice have shown that inducible nitric oxide synthase-expressing (iNOS+) inflammatory cells accumulate around lymphatic vessels. In the current study, we therefore tested the hypothesis that increased expression of iNOS results in nitrosative stress and injury to the lymphatic endothelial cells (LECs). In addition, we tested the hypothesis that lymphatic injury, independent of obesity, can modulate glucose and lipid metabolism. METHODS: We compared the metabolic changes and lymphatic function of wild-type and iNOS knockout mice fed a normal chow or high-fat diet for 16 weeks. To corroborate our in vivo findings, we analyzed the effects of reactive nitrogen species on isolated LECs. Finally, using a genetically engineered mouse model that allows partial ablation of the lymphatic system, we studied the effects of acute lymphatic injury on glucose and lipid metabolism in lean mice. RESULTS: The mesenteric lymphatic vessels of obese wild-type animals were dilated, leaky, and surrounded by iNOS+ inflammatory cells with resulting increased accumulation of reactive nitrogen species when compared with lean wild-type or obese iNOS knockout animals. These changes in obese wild-type mice were associated with systemic glucose and lipid abnormalities, as well as decreased mesenteric LEC expression of lymphatic-specific genes, including vascular endothelial growth factor receptor 3 (VEGFR-3) and antioxidant genes as compared with lean wild-type or obese iNOS knockout animals. In vitro experiments demonstrated that isolated LECs were more sensitive to reactive nitrogen species than blood endothelial cells, and that this sensitivity was ameliorated by antioxidant therapies. Finally, using mice in which the lymphatics were specifically ablated using diphtheria toxin, we found that the interaction between metabolic abnormalities caused by obesity and lymphatic dysfunction is bidirectional. Targeted partial ablation of mesenteric lymphatic channels of lean mice resulted in increased accumulation of iNOS+ inflammatory cells and increased reactive nitrogen species. Lymphatic ablation also caused marked abnormalities in insulin sensitivity, serum glucose and insulin concentrations, expression of insulin-sensitive genes, lipid metabolism, and significantly increased systemic and mesenteric white adipose tissue (M-WAT) inflammatory responses. CONCLUSIONS: Our studies suggest that increased iNOS production in obese animals plays a key role in regulating lymphatic injury by increasing nitrosative stress. In addition, our studies suggest that obesity-induced lymphatic injury may amplify metabolic abnormalities by increasing systemic and local inflammatory responses and regulating insulin sensitivity. These findings suggest that manipulation of the lymphatic system may represent a novel means of treating metabolic abnormalities associated with obesity.

[Laparoscopic radical resection of rectal cancer with preservation of the left colic artery: anatomical basis and surgical experience].

In order to increase the blood supply of anastomosis, surgeons choose to preserve the left colon artery (LCA) during the laparoscopic radical resection of rectal cancer. However, surgeons are always ailed by hemorrhage and incompletely dissection of No. 253 lymph nodes. One reason is the shortage of understanding the relationship between inferior mesenteric artery (IMA), LCA, and inferior mesenteric vein before surgery. Another reason is that surgeon always remove the lymph nodes around LCA, while don't normatively resect No. 253 lymph nodes, which affect the overall survival rate. Therefore, the "medial-to-lateral approach" for laparoscopic preservation with LCA radical resection in rectal cancer was suggested in this article. The CT technique could be used to analyze the IMA classification, which contribuated to the standard conservation of LCA. Laparoscopic radical resection of rectal cancer could be completed of high quality, through accurate definition and exactly dissection of the No. 235 lymph nodes.

Transdermal entry of yeast components elicits transient B cell-associated responses in skin-draining lymph nodes.

Immune responses to non-pathogenic yeasts induced within the draining lymph node remain to be understood. In this study, we have investigated the changes in lymphocytes and their activity in skin-draining lymph nodes in response to transdermally injected zymosan (component of the yeast cell wall). Zymosan elicited the transient increase of B cell number and activation status without affecting the capacity for proliferation. The increased B cell content in the regional lymph nodes was likely due to the reduction of B cell egress from the tissue and in part the increase of homing from the circulation. Zymosan also upregulated the inflammatory cytokines, such as IL-1ß, IL-6, IL-12, and IFNγ, regulatory cytokines IL-10 and TGFß, and lymphoid chemokine CXCL13. Among these, the expression of IL-12 and IL-10 was markedly high in B cells. Altogether, these findings demonstrate a unique B cell-associated response to non-pathogenic yeast component in the draining lymph nodes. This will provide insights into the clinical and healthcare applications of non-pathogenic beneficial microbes.

Blocking Lymph Flow by Suturing Afferent Lymphatic Vessels in Mice.

Lymphatic vessels are critical in maintaining tissue fluid balance and optimizing immune protection by transporting antigens, cytokines, and cells to draining lymph nodes (LNs). Interruption of lymph flow is an important method when studying the function of lymphatic vessels. The afferent lymphatic vessels from the murine footpad to the popliteal lymph nodes (pLNs) are well-defined as the only routes for lymph drainage into the pLNs. Suturing these afferent lymphatic vessels can selectively prevent lymph flow to the pLNs. This method allows for interference in lymph flow with minimal damage to the lymphatic endothelial cells in the draining pLN, the afferent lymphatic vessels, as well as other lymphatic vessels around the area. This method has been used to study how lymph impacts high endothelial venules (HEV) and chemokine expression in the LN, and how lymph flows through the adipose tissue surrounding the LN in the absence of functional lymphatic vessels. With the growing recognition of the importance of lymphatic function, this method will have broader applications to further unravel the function of lymphatic vessels in regulating the LN microenvironment and immune responses.

The effect of surgery on lymphoscintigraphy drainage patterns from the canine brachium in a simulated tumor model.

OBJECTIVE: To determine the effect of surgery on lymphoscintigraphy drainage patterns from the canine brachium. STUDY DESIGN: Experimental study. ANIMALS: Eight healthy research beagles. METHODS: A predefined area of skin measuring 2 × 1.5 cm in dimension was designated on either the right or left brachium. Preoperative lymphoscintigraphy was performed with technetium sulfur colloid injected into the subcutaneous tissues around the predefined anatomic location in a four-quadrant technique. Dogs underwent surgery for excision of the predefined area of skin, subcutis, and fascia of the lateral head of the triceps muscle with 1-cm margins. Eighteen days after surgery, lymphoscintigraphy was again performed with technetium sulfur colloid injected into the subcutaneous tissues around the surgical scar in a four-quadrant technique. RESULTS: Sentinel lymph nodes were identified in eight of eight dogs preoperatively and in eight of eight dogs postoperatively. Agreement between the results of the preoperative and postoperative lymphoscintigraphy studies was identified as complete in four of eight dogs and partial in four of eight dogs. Sentinel lymph node identification occurred immediately in three of eight dogs preoperatively and in eight of eight dogs postoperatively. CONCLUSION: Sentinel lymph node identification occurred faster postoperatively. Agreement or partial agreement between the results of the preoperative and postoperative lymphoscintigraphy studies was observed in eight of eight dogs. CLINICAL SIGNIFICANCE: Surgery appears to have an effect on lymphoscintigraphy drainage patterns. Additional studies are required to compare preoperative and postoperative sentinel lymph node mapping patterns in tumor-bearing dogs. However, this study provides preliminary information regarding the effect of surgery on sentinel lymph node identification.

Morphological analysis of aqueous humor drainage using QD nanoparticles and indocyanine green.

This study represents the first morphological description of the lymphatic drainage of the ciliary body in vivo by comparative hyperspectral fluorescence imaging techniques of Quantum Qdot655 (QD) nanoparticles and indocyanine green (ICG). A volume of 1.25 µl of QD was injected into the left anterior camera of all rats. Similarly,1.25 µl of ICG diluted at a ratio of one-fourth with physiological saline solution was injected into the right anterior camera of all rats. The thickness of the skin in the mandibular area, connective tissue, and the depth of the mandibular lymph node (MLN) made image retrieval difficult. For QD, 302 nm UV excitation and 605 nm fluorescence peak emission were applied. The detection of QD and ICG used in this study in the MLNs is definitive evidence that aqueous humor (AH) follows a uveolymphatic pathway. Scanning electron microscope and the energy dispersive X-ray analyzer spectrum were used to examine both the Schlemm's canal and the MLN. For the first time, the QD was detected in the cortex of MLN. The QCM analysis of both QD-AH and ICG-AH was used to determine whether there was any interaction between them. This comparative study shows the importance of experimental animal modeling in pharmacological studies regarding eye research and drugs. In a female rat, the signal was taken from the parotid lymph node with QD injections.

Efficient homing of T cells via afferent lymphatics requires mechanical arrest and integrin-supported chemokine guidance.

Little is known regarding lymph node (LN)-homing of immune cells via afferent lymphatics. Here, we show, using a photo-convertible Dendra-2 reporter, that recently activated CD4 T cells enter downstream LNs via afferent lymphatics at high frequencies. Intra-lymphatic immune cell transfer and live imaging data further show that activated T cells come to an instantaneous arrest mediated passively by the mechanical 3D-sieve barrier of the LN subcapsular sinus (SCS). Arrested T cells subsequently migrate randomly on the sinus floor independent of both chemokines and integrins. However, chemokine receptors are imperative for guiding cells out of the SCS, and for their subsequent directional translocation towards the T cell zone. By contrast, integrins are dispensable for LN homing, yet still contribute by increasing the dwell time within the SCS and by potentially enhancing T cell sensing of chemokine gradients. Together, these findings provide fundamental insights into mechanisms that control homing of lymph-derived immune cells.

A novel role for lipoxin A4 in driving a lymph node-eye axis that controls autoimmunity to the neuroretina.

The eicosanoid lipoxin A4 (LXA4) has emerging roles in lymphocyte-driven diseases. We identified reduced LXA4 levels in posterior segment uveitis patients and investigated the role of LXA4 in the pathogenesis of experimental autoimmune uveitis (EAU). Immunization for EAU with a retinal self-antigen caused selective downregulation of LXA4 in lymph nodes draining the site of immunization, while at the same time amplifying LXA4 in the inflamed target tissue. T cell effector function, migration and glycolytic responses were amplified in LXA4-deficient mice, which correlated with more severe pathology, whereas LXA4 treatment attenuated disease. In vivo deletion or supplementation of LXA4 identified modulation of CC-chemokine receptor 7 (CCR7) and sphingosine 1- phosphate receptor-1 (S1PR1) expression and glucose metabolism in CD4+ T cells as potential mechanisms for LXA4 regulation of T cell effector function and trafficking. Our results demonstrate the intrinsic lymph node LXA4 pathway as a significant checkpoint in the development and severity of adaptive immunity.