Transport and distribution of α-tocopherol in lymph,serum and liver cells in rats

Rats were cannulated in the major mesenteric lymph duct and given an intraduodenal bolus of unlabeled and α-[³H]tocopherol, and [¹⁴C]oleic acid in soybean oil. The appearance of α-tocopherol in lymph was negligible during the first 2 h and peaked 4–15 h after feeding, whereas no detectable amount was recovered in the portal vein. Intestinal absorption via the lymphatic pathway was 15.4 ± 8.9% (n = 10) and 45.9 ± 10.8% (n = 4) for α-tocopherol and [¹⁴C]oleic acid, respectively. About 99% of α-tocopherol in lymph was associated with the chylomicron fraction (d < 1.006 g/ml). In non-fasting rats, 51% of serum α-tocopherol was associated with chylomicrons/VLDL (very-low-density lipoprotein, d < 1.006 g/ml) and 47% with HDL (high-density lipoprotein, 1.05 < d < 1.21 g/ml). Our study revealed that the liver, skeletal muscle and adipose tissue contain approx. 92% of the total mass of α-tocopherol measured in ten different organs. Parenchymal and nonparenchymal liver cells contributed to 75% and 25% of the total mass of α-tocopherol in the liver, respectively.     

Do parathyroid glands from individuals of different age and gender contain lymph vessels?

Whereas lymph vessels in some endocrine glands have been thoroughly investigated, data on these vessels in human parathyroid glands are often contradictory and deficient in available literature. Therefore, the aim of this study was to histomorphologically investigate whether lymph vessels could be found in human parathyroid glands postnatally and, if so, whether their presence was age- and gender-dependent. A total of 44 parathyroid glands from subjects of both genders, aged 4-90 years, were studied. The glands were divided into three groups. Those from the 1st and the 2nd age group demonstrated similar morphological structure of parenchyma with predominant chief cells with pale-staining cytoplasm, while the frequency of lymph vessels was lower in the 2nd group. Unlike in these groups, chief cells with dark- staining cytoplasm predominated in the glandular parenchyma of the 3rd age group where lymph vessels were not found in any of the examined glands. The frequency of lymph vessels in parathyroid glands was almost the same for both genders. Histomorphologic occurrence of lymph vessels coincided with the presence of endocrine cells with pale-staining cytoplasm, which allowed the assumption that lymph vessels were also one of the signs of functional activity of human parathyroid glands.     

Is Stereotactic Body Radiotherapy (SBRT) in lymph node oligometastatic patients feasible and effective?

ObjectivesTo review the available data about stereotactic body-radiotherapy (SBRT) for oligometastatic lymph node cancer recurrence.MethodsThe inclusion criteria for this study were as follows: Medline search for the (1) English language (2) full paper (abstracts were excluded) on (3) adult oligometastatic solid cancer recurrence limited to lymph node that underwent SBRT (4) outcome data available and (5) published up to the 30th April 2014.Results38 papers fulfilling the inclusion criteria have been found: 7 review articles and 31 patient series (20 and 11 retrospective and prospective studies, respectively) including between 1 and 69 patients (636 lymph nodes). Twelve articles reported only lymph node SBRT while in 19 – all types of SBRT including lymph node SBRT were presented. Two-year local control, 4-year progression free survival and overall survival was of up to 100%, 30% and 50%, respectively. The progression was mainly out-field (10–30% of patients had a recurrence in another lymph node/nodes). The toxicity was low with mainly mild acute events and single grade 3–4 late events. When compared to SBRT for any oligometastatic cancer, SBRT for lymph node recurrence carried better prognosis and showed lower toxicity.ConclusionsSBRT is a feasible approach for oligometastatic lymph node recurrence, offering excellent in-field tumor control with low toxicity profile. The potential abscopal effect has been hypothesized as a basis of these findings. Future studies are warranted to identify the patients that benefit most from this treatment. The optimal combination with systemic treatment should also be defined.     

γδ T cell homing to skin and migration to skin-draining lymph nodes is CCR7 independent

In most species, γδ T cells preferentially reside in epithelial tissues like the skin. Lymph duct cannulation experiments in cattle revealed that bovine dermal γδ T cells are able to migrate from the skin to the draining lymph nodes via the afferent lymph. For αβ T cells, it is generally accepted that epithelial and mucosal tissue egress is regulated by expression of the CCR7 chemokine receptor. In this study, we tracked the migratory route of bovine lymph-derived γδ T cells and examined their CCR7 cell surface expression in several compartments along this route. Total lymph cells from afferent and efferent origin were labeled with PKH fluorescent dyes and injected into the bloodstream. PKH(+) cells already reappeared in the afferent lymph after 4 h. The vast majority of the PKH(+) cells retrieved from the afferent lymph were of the WC1(+) γδ T cell phenotype, proving that this PKH(+) γδ T cell subset is able to home to and subsequently exit the skin. PKH(+) γδ T cells from afferent and efferent lymph lack CCR7 surface expression and display high levels of CD62L compared with CD4 T cells, which do express CCR7. Skin homing receptors CCR4 and CCR10 in contrast were transcribed by both CD4 and γδ T cells. Our findings suggest that γδ T cell skin egress and migration into the peripheral lymphatics is CCR7-independent and possibly mediated by CD62L expression.     

Interdigitating reticulum cells in lymph nodes of Sézary syndrome. Freeze-fracture and ultrathin-section morphology

Lymph nodes with extensive leukemic infiltration from three patients with the Sézary syndrome were examined in ultrathin sections and in freeze-fracture replicas. Sézary cells (SC) and interdigitating reticulum cells (IDC) were the predominant cell types in the lymph nodes. Both were closely connected with each other by apparently interdigitating cytoplasmic processes. The projections between these cells were, in the main, processes from the IDC. In freeze-fracture replicas these cellular processes did not appear as interdigitations but were more bubble-like, and for this reason these cells are imprecisely described by the term "interdigitating." The SC were seen to possess only short cytoplasmic processes. The frequent polar grouping of cell organelles in SC in the region of the contact zone with IDC and the high organelle content of IDC ('activated IDC') could be the morphologic expression of intense interaction between IDC and SC. IDC displayed three features in freeze-fracture which are not specific to the Sézary syndrome, but should be applicable to IDC in general: (1) they exhibited an approximately equal density of intramembrane particles in both the E-face and the P-face, (2) some of the intramembrane particles in the P-face were assembled in clusters and (3) the surface showed bubble-like formations of the cytoplasmic processes. On the basis of these properties it was possible to distinguish IDC from macrophages and lymphocytes in freeze-fracture replicas.     

Radiomic models for lymph node metastasis prediction in cervical cancer: can we think beyond sentinel lymph node?

Liu and colleagues performed a retrospective study to validate a computed tomography (CT) scan-based radiomic model to detect lymph node metastasis in cervical cancer. The proposed model incorporating the arterial and venous phase CT-scan features represented a non-invasive method exhibiting high sensitivity in the prediction of lymph node metastasis. It is well established that lymph node metastasis is one of the most significant prognostic factors in cervical cancer. For this reason, management of cervical cancer is strictly related to lymph node status, with international guidelines recommending definitive chemo-radiation in case of metastatic lymph node. More and more evidence supports the use of sentinel lymph node in early-stage cervical cancer but its frozen section analysis may result in false negative results; in locally-advanced stages staging para-aortic lymphadenectomy is proposed by many Authors to tailor chemoradiotherapy treatment, with potential intra-and post-operative related complications. The use of a validated radiomic model able to predict lymph node metastases in radiologically normal lymph nodes may represent an essential tool to possibly spare lympadenectomy related morbidity.     

Increased secretion of insulin and proliferation of islet β-cells in rats with mesenteric lymph duct ligation

Background &; aimsIt has been suggested that intestinal lymph flow plays an important role in insulin secretion and glucose metabolism after meals. In this study, we investigated the influence of ligation of the mesenteric lymph duct on glucose metabolism and islet β-cells in rats.MethodsMale Sprague–Dawley rats (10 weeks old) were divided into two groups: one underwent ligation of the mesenteric lymph duct above the cistern (ligation group), and the other underwent a sham operation (sham group). After 1 and 2 weeks, fasting plasma concentrations of glucose, insulin, triglyceride, glucose-dependent insulinotropic polypeptide (GIP), and the active form of glucagon-like peptide-1 (GLP-1) were measured. At 2 weeks after the operation, the oral glucose tolerance test (OGTT) and intravenous glucose tolerance test (IVGTT) were performed. After the rats had been sacrificed, the insulin content of the pancreas was measured and the proliferation of β-cells was assessed immunohistochemically using antibodies against insulin and Ki-67.ResultsDuring the OGTT, the ligation group showed a significant decrease in the plasma glucose concentration at 120 min (p < 0.05) and a significant increase in the plasma insulin concentration by more than 2-fold at 15 min (p < 0.01). On the other hand, the plasma GIP concentration was significantly decreased at 60 min (p < 0.01) in the ligated group, while the active form of GLP-1 showed a significantly higher level at 90 min (1.7-fold; p < 0.05) and 120 min (2.5-fold; p < 0.01). During the IVGTT, the plasma insulin concentration in the ligation group was significantly higher at 2 min (more than 1.4-fold; p < 0.05). Immunohistochemistry showed that the ratios of β-cell area/acinar cell area and β-cell area/islet area, and also β-cell proliferation, were significantly higher in the ligation group than in the sham group (p < 0.05, p < 0.01 and p < 0.01, respectively). The insulin content per unit wet weight of pancreas was also significantly increased in the ligation group (p < 0.05).ConclusionsIn rats with ligation of the mesenteric lymph duct, insulin secretion during the OGTT or IVGTT was higher, and the insulin content and β-cell proliferation in the pancreas were also increased. Our data show that mesenteric lymph duct flow has a role in glucose metabolism.     

Analysis of T cell receptor β and γ genes from peripheral blood,regional lymph node and tumor-infiltrating lymphocyte clones from melanoma patients

Summary A total of 199 T cell clones from two melanoma patients were derived from progenitor T cells from recurrent melanoma, regional lymph nodes (either involved or uninvolved with malignancy) and peripheral blood by inoculating single cells directly into the wells of microtiter plates before in vitro expansion. The surface marker phenotype of most clones was CD4⁺CD8^–, although some were CD4^–CD8⁺. Genomic DNA prepared from all clones was analyzed by Southern blot hybridization using T cell receptor (TCR) and gene probes, seeking clones with identical TCR gene rearrangement patterns as direct evidence for in vivo progenitor T cell clonal amplification. ProbingHindIII-digested DNA with TCR and TCR probes revealed several clones with identical TCR gene rearrangement patterns. These clones had subsequent probing ofBamHI-digested DNA with TCR and TCR probes, which showed all but 2 clones to have distinct rearrangement patterns. These analyses provide clear molecular evidence for in vivo polyclonal CD4⁺ T cell populations in each of several separate immune compartments in these patients.This investigation was supported by National Institutes of Health, National Research Service Award CA-08 397 from the National Cancer Institute as well as NIH CA-32 685, CA-30 688, DOE FG028 760 502 and American Cancer Society Grant ACS CH-237     

Mesenteric lymph system constitutes the second route in gut–liver axis and transports metabolism-modulating gut microbial metabolites

The gut–liver axis denotes the intricate connection and interaction between gut microbiome and liver, in which compositional and functional shifts in gut microbiome affect host metabolism. Hepatic portal vein of the blood circulation system has been thought to be the major route for metabolite transportation in the gut–liver axis, but the existence and importance of other routes remain elusive. Here, we perform metabolome comparison in blood circulation and mesenteric lymph systems and identify significantly shifted metabolites in serum and mesentery. Using cellular assays, we find that the majority of decreased metabolites in lymph system under high-fat diet are effective in alleviating metabolic disorders, indicating a high potential of lymph system in regulating liver metabolism. Among those, a representative metabolite, L-carnitine, reduces diet-induced obesity in mice. Metabolic tracing analysis identifies that L-carnitine is independently transported by the mesenteric lymph system, serving as an example that lymph circulation comprises a second route in the gut–liver axis to modulate liver metabolism. Our study provides new insights into metabolite transportation via mesenteric lymph system in the gut–liver axis, offers an extended scope for the investigations in host-gut microbiota metabolic interactions and potentially new targets in the treatment of metabolic disorders.     

Expression of TGF-β1, SNAI1 and MMP-9 is associated with lymph node metastasis in papillary thyroid carcinoma

TGF-β1, SNAI1 and MMP-9 are implicated in tumor invasion and metastasis. The purpose of this study was to examine TGF-β1, SNAI1 and MMP-9 expression in papillary thyroid carcinoma (PTC), and to assess association of TGF-β1, SNAI1 and MMP-9 expression with several clinicopathological indicators of PTC. TGF-β1, SNAI1 and MMP-9 protein expression in 83 PTCs and their matched normal thyroid specimens were analyzed using immunohistochemistry. The mRNA expression levels of TGF-β1, SNAI1 and MMP-9 in 12 fresh PTC specimens with lymph node metastasis (LNM), 12 fresh PTC specimens without LNM and their matched normal thyroid specimens were assessed by real-time RT-PCR. The results showed that the mRNA and protein expression levels of TGF-β1, SNAI1 and MMP-9 were significantly higher in PTCs than in their matched normal thyroid tissues. There were not significant differences in TGF-β1, SNAI1 and MMP-9 protein expression relative to age, gender, tumor size and TNM stage, except for MMP-9 whose protein expression correlated with tumor size. However, high mRNA and protein expression levels of TGF-β1, SNAI1 and MMP-9 were significantly correlated with LNM. Furthermore, TGF-β1, SNAI1 and MMP-9 protein expression were significantly correlated with one another. Concomitant expression of any two or all of the three molecules had stronger correlation with LNM than did each alone. Collectively, the present results indicate that immunohistochemical and real-time RT-PCR evaluation of TGF-β1, SNAI1 and MMP-9 expression in PTC may be useful to predict the risk of LNM in PTC patients.     

PTGS2 (COX-2) −765 G > C functional promoter polymorphism and its association with risk and lymph node metastasis in nasopharyngeal carcinoma

Cyclooxygenase-2 (Cox-2) is a key enzyme in the conversion of arachidonic acid to prostaglandins that has been shown to have a particular importance in the progression of several malignancies including nasopharyngeal carcinoma (NPC). In the current report, we designed a case-controlled study to evaluate the susceptibility and prognostic implications of the functional −765 G > C genetic variation in NPC. A PCR and restriction fragment length polymorphism analysis was used to determine the polymorphism in a Tunisian population of patients with NPC (n = 180) and in healthy control subjects (n = 169). A higher risk for NPC was observed for carriers of COX-2 −765 C allele (OR = 1.76; P = 0.01). This association remains significant after adjustments for age and sex (OR = 1.89; P = 0.008). Regarding prognostic indicators, a significant association was found between −765 C allele carriers and the presence of lymph node metastasis (OR = 2.28; P = 0.01), as well as, with tumor stage (OR = 2.73; P = 0.03). This is the first report on the studies of COX-2 SNPs in NPC and our data suggest that this genetic variant may play a role in mediating susceptibility to NPC, as well as, in neoplastic progression, a finding which further supports the involvement of COX-2 in NPC etiology. Hela Ben Nasr and Karim Chahed contributed equally to the study.     

Caveolin-1 overexpression is associated with simultaneous abnormal expression of the E-cadherin/α-β catenins complex and multiple ErbB receptors and with lymph nodes metastasis in head and neck squamous cell carcinomas

The presence of lymph node metastases is one of the most important prognostic indicators in head and neck squamous cell carcinomas (HNSCCs). An alteration of the E-cadherin-catenins complex and EGFR is essential for the invasiveness of cancer cells. Caveolin-1, the major structural protein of the caveolae, represents a scaffolding molecule for several signaling proteins including EGFR. Although caveolin-1 has been shown to play a role in inducing the invasive phenotype of cancer cells, its role appears to be cell-type specific and for some tumors it has not been defined yet. In this study we used 57 HNSCC specimens to investigate whether the abnormal expression of caveolin-1 was associated with the derangement of the E-cadherin-catenins complex and with the overexpression of ErbB receptors. We demonstrate that in HNSCCs caveolin-1 overexpression is associated with the simultaneous abnormal expression of at least one member of the E-cadherin/α-β catenins complex and multiple ErbB receptors as well as with lymph node metastases. We also demonstrate that chronic stimulation of a human hypopharyngeal carcinoma cell line (FaDu) with EGF induced the internalization of β-catenin and caveolin-1 and their co-localization with EGFR. Moreover, EGF treatment induced an increased physical interaction between EGFR/β-catenin/caveolin-1 and between E-cadherin/β-catenin/caveolin-1. These molecular events were associated with an increased directional motility of FaDu cells in vitro. These findings may provide new insight into the biology of HNSCC progression and help to identify subgroups of primary HNSCCs with a more aggressive behavior.     

Mitochondrial metabolism in cancer metastasis: Visualizing tumor cell mitochondria and the “reverse Warburg effect” in positive lymph node tissue

We have recently proposed a new two-compartment model for understanding the Warburg effect in tumor metabolism. In this model, glycolytic stromal cells produce mitochondrial fuels (L-lactate and ketone bodies) that are then transferred to oxidative epithelial cancer cells, driving OXPHOS and mitochondrial metabolism. Thus, stromal catabolism fuels anabolic tumor growth via energy transfer. We have termed this new cancer paradigm the “reverse Warburg effect,” because stromal cells undergo aerobic glycolysis, rather than tumor cells. To assess whether this mechanism also applies during cancer cell metastasis, we analyzed the bioenergetic status of breast cancer lymph node metastases, by employing a series of metabolic protein markers. For this purpose, we used MCT4 to identify glycolytic cells. Similarly, we used TOMM20 and COX staining as markers of mitochondrial mass and OXPHOS activity, respectively. Consistent with the “reverse Warburg effect,” our results indicate that metastatic breast cancer cells amplify oxidative mitochondrial metabolism (OXPHOS) and that adjacent stromal cells are glycolytic and lack detectable mitochondria. Glycolytic stromal cells included cancer-associated fibroblasts, adipocytes and inflammatory cells. Double labeling experiments with glycolytic (MCT4) and oxidative (TOMM20 or COX) markers directly shows that at least two different metabolic compartments co-exist, side-by-side, within primary tumors and their metastases. Since cancer-associated immune cells appeared glycolytic, this observation may also explain how inflammation literally “fuels” tumor progression and metastatic dissemination, by “feeding” mitochondrial metabolism in cancer cells. Finally, MCT4(+) and TOMM20(-) “glycolytic” cancer cells were rarely observed, indicating that the conventional “Warburg effect” does not frequently occur in cancer-positive lymph node metastases.Key words: caveolin-1, oxidative stress, MCT4, metabolic coupling, tumor stroma, SLC16A3, monocarboxylic acid transporter, two-compartment tumor metabolism, metastasis, TOMM20, complex IV, OXPHOS, mitochondria, inflammation     

Upregulation of microRNA-141 suppresses epithelial-mesenchymal transition and lymph node metastasis in laryngeal cancer through HOXC6-dependent TGF-β signaling pathway

Laryngeal cancer is one of the most malignant cancers among the head and neck malignant tumors. Abnormal expression of microRNAs (miRNAs) contributes to cancer development through regulating proliferation and apoptosis of cancer cells. In this study, we aim to explore the roles of microRNA-141 (miR-141), Homeobox C6 (HOXC6) and TGF-β signaling pathway in epithelial-mesenchymal transition (EMT) and lymph node metastasis in laryngeal cancer. Initially, we identified differentially expressed genes in laryngeal cancer, among which HOXC6 was identified. Then the target miRNA of HOXC6 was predicted and verified. Next, expression of miR-141, HOXC6, TGF-β1, Smad3, Vimentin and Snail in cancer tissues was detected. Then, AMC-HN-8 cells were transfected with miR-141 mimic, miR-141 inhibitor and HOXC6-siRNA to investigate specific role of miR-141, HOXC6 and TGF-β signaling pathway in laryngeal cancer in vivo and in vitro. Our results showed that HOXC6 was a target gene of miR-141, which was downregulated in laryngeal cancer. Besides, overexpression of miR-141 could downregulate HOXC6 and inhibit the TGF-β signaling pathway. Upregulation of miR-141 or silencing of HOXC6 can repress EMT, viability, migration and invasion abilities of laryngeal cancer cells. In addition, upregulation of miR-141 inhibited the tumor growth and lymph node metastasis in vivo. In summary, our findings demonstrated that upregulated miR-141 decreased HOXC6 expression, and inhibited the TGF-β signaling pathway, EMT and lymph node metastasis in laryngeal cancer, which is of clinical significance in the treatment of laryngeal cancer.     

Tumor-specific granulocyte/macrophage colony-stimulating factor and interferon γ secretion is associated with in vivo therapeutic efficacy of activated tumor-draining lymph node cells

In this study, cytokine release by tumor-draining lymph node cells sensitized in vitro (IVS-TDLN) was examined and correlated with therapeutic efficacy in adoptive immunotherapy. Mice bearing immunologically distinct MCA 207 and MCA 205 sarcoma tumors were utilized in criss-cross experiments. IVS-TDLN obtained from mice bearing 10-day subcutaneous (s. c.) tumors mediated immunologically specific regression of established 3-day pulmonary metastases, but demonstrated non-specific cytolytic reactivity against both tumors in a 4-h⁵¹Cr-release assay. By contrast, these IVS-TDLN cells were found specifically to secrete granulocyte/macrophage colony-stimulating factor (GM-CSF) and interferon (IFN) when restimulated in vitro with irradiated tumor cells. To determine the predictive value of tumor-specific cytokine release with in vivo therapeutic efficacy, a kinetic analysis of antitumor activities of TDLN obtained from animals bearing MCA 207 tumors for increasing lengths of time was performed. IVS-TDLN cells from mice bearing day-7, -10 and-14 s. c. tumors manifested tumor-specific release of GM-CSF and IFN, and mediated significant antitumor reactivity in vivo. In contrast IVS-LN cells from day-0 and day-21 tumor-bearing animals did not release significant amounts of GM-CSF and IFN, and were not therapeutically efficacious in vivo. Day-4 IVS-TDLN released high levels of GM-CSF and IFN non-specifically, and were not therapeutic in adoptive immunotherapy at doses effective for day-7 and day-14 IVS-TDLN cells. In other experiments, IVS cells generated from different lymph node groups in animals bearing 10-day established s. c. tumors were examined and found to have unique profiles of cytokine release. In these studies, the ability of IVS cells to release specifically both cytokines as opposed to one was associated with greater therapeutic efficacy on a per cell basis. Our findings suggest that the tumor-specific releases of GM-CSF and IFN are useful parameters to assess the in vivo therapeutic efficacy of immune lymphocytes.