High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 µm, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse  model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological  analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.     

Primary Tumor-Secreted Lymphangiogenic Factors Induce Pre-Metastatic Lymphvascular Niche Formation at Sentinel Lymph Nodes in Oral Squamous Cell Carcinoma

Objectives

The objectives of this study were to evaluate the formation of lymphvascular niches in lymph nodes of patients with oral squamous cell carcinoma (OSCC), and investigate the roles of lymphangiogenic and angiogenic factors, such as vascular endothelial growth factor (VEGF)-A, VEGF-C, and VEGF-D, expressed in the primary tumors.

Materials and Methods

Forty-four patients with previously untreated clinically late T2 or T3 OSCC of cN0 were evaluated for primary tumors and 166 sentinel lymph nodes (SLNs). Primary tumors were immunohistochemically analyzed for expressions of VEGFs. Densities of lymphatic vessels (LVD_podoplanin) and high endothelial venules (HEVD) in the SLNs were also calculated using antibodies for each marker, podoplanin and MECA-79, respectively.

Results

In 25 patients, all lymph nodes were metastasis-negative, whereas, in 19 patients, metastasis was positive for at least one lymph node (either at SLN, non-SLN, or nodal recurrence). From the analyses of 140 SLNs without metastasis, LVD_podoplanin in 50 SLNs of metastasis-positive cases was significantly higher than that in 90 SLNs of metastasis-negative cases (p = 0.0025). HEVD was not associated with lymph node metastasis. The patients with VEGF-A-High or VEGF-D-High tumors had significantly higher LVD_podoplanin than patients with their Low counterparts (p = 0.0233 and p = 0.0209, respectively). In cases with lymph node metastasis, the VEGF-D-expression score was significantly higher than in those without lymph node metastasis (p = 0.0006).

Conclusions

These results suggest that lymph node lymphangiogenesis occurs before metastasis in OSCC. VEGF-A and VEGF-D play critical roles in this process. VEGF-D is a potential predictive marker of positive lymph node metastasis in cN0 patients.     

The Selective Staining of Curious Bodies in Lymph Nodes of Patients as a Means for Diagnosis of Sarcoid

Paraffin sections of formalin-fixed tissue from some human lymph nodes manifesting the characteristic lesions of sarcoid contain minute bodies 3-15 μ in diameter. These bodies can be stained by direct application -of Schiff's reagent adjusted to pH 3-3.5, without prior hydrolysis or oxidation. They react positively to the naphthoic acid hydrazide procedure for aldehyde, and their reactivity to both these reagents was blocked by aniline and dimedone. These reactions indicate that the peculiar bodies in sarcoid tissues contain reactive aldehydes, present either before or after the processing of the tissue.     

Optimal Extent of Prophylactic Irradiation of Paraaortic Lymph Nodes in Patients with Uterine Cervical Cancer

Purpose

The purpose of this study was to determine optimal extent of prophylactic irradiation of paraaortic lymph nodes (PALN) in patients with uterine cervical cancer who had metastatic pelvic LNs.

Methods and Materials

We retrospectively evaluated 103 patients with cervical cancer and pelvic lymph node metastasis who were treated with prophylactic semi-extended field radiotherapy (SEFRT) between 1990 and 2012. The semi-extended field included PALN below the second lumbar spine with prescribed doses of 45 to 50 Gy. Survival outcomes were calculated using the Kaplan-Meier method, and acute and late toxicities were scored using the Radiation Therapy Oncology Group and European Organisation for Research and Treatment of Cancer toxicity criteria.

Results

The median follow-up after SEFRT was 61 (range 5–296) months. Overall, 28 patients (27.2%) experienced treatment failures, which were classified as local in 8 patients (7.8%), regional in 8 patients (7.8%), and distant in 13 patients (12.6%). Of the regional failures, only two involved PALN failure around the renal artery or the renal hilum area at the upper margin of the semi-extended field. At 5 years, the overall survival was 82%. Grade 3 or higher acute gastrointestinal and genitourinary toxicities occurred in one and two patients, respectively. As a late toxicity, one patient developed grade 3 small bowel obstruction.

Conclusion

Prophylactic SEFRT provided favorable outcomes with little acute or late gastrointestinal toxicity. For prophylaxis of PALN recurrences, upper part of PALN might not need to be included in patients with uterine cervical cancer and metastatic pelvic LNs.     

Prospects for Vasculature Reorganization in Sentinel Lymph Nodes

Primary tumors can induce vasculature and lymph channel reorganizations within sentinel lymph nodes before the arrival of cancer cells. The key blood vessels in such nodes that are remodeled are high endothelial venules (HEVs). The morphological alteration of HEVs in the presence of a cancer, coupled with the increased proliferation rate of the endothelial cells, results in a functional shifting of HEVs from immune response to blood-flow carrier. This tumor-induced reorganization is quite different from an endotoxin-induced inflammatory vasculature alteration. We review some of the accepted doctrines on lymph flow and lymphatic metastasis in light of this reorganization. More investigations are needed to elucidate the molecular mechanisms underlying the morphological and functional alteration of HEVs, the fluid exchanges between lymph and blood, the microenvironmental preparation for cancer cells to survive and expand in the lymph node, and the detail process of further distant dissemination of cancer cells from the lymph nodes. Further study of this pathological process may help to explain some clinical phenomena, and should aid in developing novel therapeutics and prevention strategies against cancer metastasis.     

磁感应热疗联合放疗治疗颈部淋巴结复发的临床观察

目的：探索磁感应热疗技术联合放疗治疗不宜手术的颈部淋巴结复发的疗效和安全性。方法：2012年2月至2012年7月选取27例不宜手术的颈部淋巴结复发病例,给予2次磁热籽植入和4—8次不等的加热治疗,计划治疗目标温度为48—50℃、维持30min,磁感应治疗后一周内行放射治疗,治疗结束后1—3个月时复查CT,治疗后6月内不予化疗。结果：治疗结束后3个月时,27例患者中CR55．6％,PR37％,NC3．7％,PD3．7％,治疗总有效率为92．6％;25例疼痛缓解,疼痛缓解有效率92．6％。治疗结束后6个月未发现远处转移病例,未发生大出血、放射性食管炎、放射性骨坏死、骨髓抑制、粒子移位脱落等并发症。结论：磁感应热疗联合放疗治疗不宜手术的复发颈部淋巴结安全、有效。     

Lymphocytic Thyroiditis is Associated with Increased Number of Benign Cervical Nodes and Fewer Central Neck Compartment Metastatic Lymph Nodes in Patients with Differentiated Thyroid Cancer

Objective: Whether or not autoimmune thyroid disease influences the progression of differentiated thyroid cancer (DTC) remains controversial. Findings of previous studies are influenced by lead time bias and/or procedure bias selection. These biases can be reduced by studying a single-institution patient population that underwent a similar extent of surgical resection.Methods: From a cohort of 660 patients with DTC who underwent thyroidectomy, we retrospectively studied 357 patients who underwent total thyroidectomy and central compartment node dissection (CCND) for DTC between 2003 and 2013.Results: Forty-one percent (140/345) of study patients had lymphocytic thyroiditis (LT), and 30% (91/301) had serum positive for thyroglobulin antibody (TgAb). LT was reported in 78% of the TgAb-positive cases. Sixty percent (213/357) of cases had metastatic thyroid carcinoma in 1 or more neck lymph nodes (55% [198/357] central compartment, and 22% [77/356] lateral compartment). Patients with LT had fewer metastatic cervical lymph nodes than those with no LT (2.7 ± 4.7 vs 3.5 ± 4.8, respectively, P = .0285). Patients with positive TgAb and thyroiditis had a larger number of benign cervical lymph nodes removed than those with negative TgAb or no LT. No significant difference was observed in age, tumor size, multifocality, extrathyroidal extension, vascular invasion, or frequency of cervical lymph node metastasis between TgAb-negative and -positive cases or between cases with and without LT.Conclusion: Lymphocytic thyroiditis is associated with fewer central neck compartment metastatic lymph nodes and a larger number of excised reactive benign cervical lymph nodes. Whether this association indicates a protective role of thyroid autoimmunity in lymph node spreading remains unclear.Abbreviations:CCND = central compartment node dissectionDTC = differentiated thyroid cancerHT = Hashimoto thyroiditisLT = lymphocytic thyroiditisTgAb = thyroglobulin antibodyTPO = thyroid peroxidase     

Applicability of Preoperative Nuclear Morphometry to Evaluating Risk for Cervical Lymph Node Metastasis in Oral Squamous Cell Carcinoma

Background

We previously reported the utility of preoperative nuclear morphometry for evaluating risk for cervical lymph node metastases in tongue squamous cell carcinoma. The risk for lymph node metastasis in oral squamous cell carcinoma, however, is known to differ depending on the anatomical site of the primary tumor, such as the tongue, gingiva, mouth floor, and buccal mucosa. In this study, we evaluated the applicability of this morphometric technique to evaluating the risk for cervical lymph node metastasis in oral squamous cell carcinoma.

Methods

A digital image system was used to measure the mean nuclear area, mean nuclear perimeter, nuclear circular rate, ratio of nuclear length to width (aspect ratio), and nuclear area coefficient of variation (NACV). Relationships between these parameters and nodal status were evaluated by t-test and logistic regression analysis.

Results

Eighty-eight cases of squamous cell carcinoma (52 of the tongue, 25 of the gingiva, 4 of the buccal mucosa, and 7 of the mouth floor) were included: 46 with positive node classification and 42 with negative node classification. Nuclear area and perimeter were significantly larger in node-positive cases than in node-negative cases; however, there were no significant differences in circular rate, aspect ratio, or NACV. We derived two risk models based on the results of multivariate analysis: Model 1, which identified age and mean nuclear area and Model 2, which identified age and mean nuclear perimeter. It should be noted that primary tumor site was not associated the pN-positive status. There were no significant differences in pathological nodal status by aspect ratio, NACV, or primary tumor site.

Conclusion

Our method of preoperative nuclear morphometry may contribute valuable information to evaluations of the risk for lymph node metastasis in oral squamous cell carcinoma.     

Comparison of Six Methods for Isolating Mycobacteria from Swine Lymph Nodes

Six laboratory methods were compared for isolating acid-fast bacteria. Tuberculous lymph nodes from each of 48 swine as identified by federal meat inspectors were processed by each of the methods. Treated tissue suspensions were inoculated onto each of eight media which were observed at 7-day intervals for 9 weeks. There were no statistically significant differences between the number of Mycobacterium avium complex bacteria isolated by each of the six methods. Rapid tissue preparation methods involving treatment with 2% sodium hydroxide or treatment with 0.2% zephiran required only one-third to one-fourth the processing time as a standard method. There were small differences in the amount of contamination among the six methods, but no detectable differences in the time of first appearance of M. avium complex colonies.     

GRAbB: Selective Assembly of Genomic Regions,a New Niche for Genomic Research

GRAbB (Genomic Region Assembly by Baiting) is a new program that is dedicated to assemble specific genomic regions from NGS data. This approach is especially useful when dealing with multi copy regions, such as mitochondrial genome and the rDNA repeat region, parts of the genome that are often neglected or poorly assembled, although they contain interesting information from phylogenetic or epidemiologic perspectives, but also single copy regions can be assembled. The program is capable of targeting multiple regions within a single run. Furthermore, GRAbB can be used to extract specific loci from NGS data, based on homology, like sequences that are used for barcoding. To make the assembly specific, a known part of the region, such as the sequence of a PCR amplicon or a homologous sequence from a related species must be specified. By assembling only the region of interest, the assembly process is computationally much less demanding and may lead to assemblies of better quality. In this study the different applications and functionalities of the program are demonstrated such as: exhaustive assembly (rDNA region and mitochondrial genome), extracting homologous regions or genes (IGS, RPB1, RPB2 and TEF1a), as well as extracting multiple regions within a single run. The program is also compared with MITObim, which is meant for the exhaustive assembly of a single target based on a similar query sequence. GRAbB is shown to be more efficient than MITObim in terms of speed, memory and disk usage. The other functionalities (handling multiple targets simultaneously and extracting homologous regions) of the new program are not matched by other programs. The program is available with explanatory documentation at https://github.com/b-brankovics/grabb. GRAbB has been tested on Ubuntu (12.04 and 14.04), Fedora (23), CentOS (7.1.1503) and Mac OS X (10.7). Furthermore, GRAbB is available as a docker repository: brankovics/grabb (https://hub.docker.com/r/brankovics/grabb/).

This is a PLOS Computational Biology Software paper

     

Altruists Proliferate Even at a Selective Disadvantage within Their Own Niche

The evolutionary origin of altruism is a long-standing puzzle. Numerous explanations have been proposed, most prominently based on inclusive fitness or group selection. One possibility that has not yet been considered is that new niches will be created disproportionately often when altruism appears, perhaps by chance, causing altruists to be over-represented in such new niches. This effect is a novel variant of group selection in which altruistic groups benefit by discovering unoccupied niches instead of by competing for the limited resources within a single niche. Both an analytical population genetics model and computational simulations support that altruism systematically arises due to this side effect of increased carrying capacity even when it is strongly selected against within any given niche. In fact, even when selection is very strongly negative and altruism does not develop in most populations, it can still be expected to be observed in a consistent fraction of species. The ecological structure provided by niches thereby may be sufficient for altruists to proliferate even if they are always at a disadvantage within each niche considered individually.     

Ultrasonographic Differentiation of Cervical Lymph Nodes in Patients with Papillary Thyroid Carcinoma After Thyroidectomy and Radioiodine Ablation: A Prospective Study

ObjectiveThe objective of the present study was to validate an ultrasound (US) classification of cervical lymph nodes (LNs) in patients with papillary thyroid cancer (PTC) after thyroidectomy and radioactive iodine (¹³¹I) ablation.MethodsWe performed a prospective study in which the patients were submitted to thyroidectomy and ¹³¹I ablation and then followed until neck US revealed LN(s) ≥ 5 mm. A total of 288 LNs from 112 patients with PTC were evaluated. Patient management was based on LN characteristics grouped according to the classification system studied here.ResultsThe presence of microcalcifications and/or cystic degeneration of cervical LNs were highly suggestive of a metastatic etiology (specificity of 99.4%). In contrast, the most sensitive finding for LNs affected by PTC was the absence of an echogenic hilum (sensitivity of 100%). In the absence of these findings (microcalcifications, cystic degeneration, echogenic hilum), a metastatic etiology was the most likely in the case of a round LN (specificity of 89%). The differentiation of a spindle-shaped LN without a visible hilum by Doppler analysis permitted us to dichotomize an initial probability of metastases of 13% in 25% (with peripheral vascularization) versus 3.3% (without peripheral vascularization).ConclusionsOur results confirm that the classification proposed for cervical LNs in patients with PTC is valid for determining patient management following initial therapy. (Endocr Pract. 2014;20:293-298)     

Focus: Microbiome: Distinct Ecological Niche of Anal,Oral, and Cervical Mucosal Microbiomes in Adolescent Women

Human body sites represent ecological niches for microorganisms, each providing variations in microbial exposure, nutrient availability, microbial competition, and host immunological responses. In this study, we investigated the oral, anal, and cervical microbiomes from the same 20 sexually active adolescent females, using culture-independent, next-generation sequencing. DNA from each sample was amplified for the bacterial 16S rRNA gene and sequenced on an Illumina platform using paired-end reads. Across the three anatomical niches, we found significant differences in bacterial community composition and diversity. Overall anal samples were dominated with Prevotella and Bacteriodes, oral samples with Streptococcus and Prevotella, and cervical samples with Lactobacillus. The microbiomes of a few cervical samples clustered with anal samples in weighted principal coordinate analyses, due in part to a higher proportion of Prevotella in those samples. Additionally, cervical samples had the lowest alpha diversity. Our results demonstrate the occurrence of distinct microbial communities across body sites within the same individual.     

InlA Promotes Dissemination of Listeria monocytogenes to the Mesenteric Lymph Nodes during Food Borne Infection of Mice

Intestinal Listeria monocytogenes infection is not efficient in mice and this has been attributed to a low affinity interaction between the bacterial surface protein InlA and E-cadherin on murine intestinal epithelial cells. Previous studies using either transgenic mice expressing human E-cadherin or mouse-adapted L. monocytogenes expressing a modified InlA protein (InlA^m) with high affinity for murine E-cadherin showed increased efficiency of intragastric infection. However, the large inocula used in these studies disseminated to the spleen and liver rapidly, resulting in a lethal systemic infection that made it difficult to define the natural course of intestinal infection. We describe here a novel mouse model of oral listeriosis that closely mimics all phases of human disease: (1) ingestion of contaminated food, (2) a distinct period of time during which L. monocytogenes colonize only the intestines, (3) varying degrees of systemic spread in susceptible vs. resistant mice, and (4) late stage spread to the brain. Using this natural feeding model, we showed that the type of food, the time of day when feeding occurred, and mouse gender each affected susceptibility to L. monocytogenes infection. Co-infection studies using L. monocytogenes strains that expressed either a high affinity ligand for E-cadherin (InlA^m), a low affinity ligand (wild type InlA from Lm EGDe), or no InlA (ΔinlA) showed that InlA was not required to establish intestinal infection in mice. However, expression of InlA^m significantly increased bacterial persistence in the underlying lamina propria and greatly enhanced dissemination to the mesenteric lymph nodes. Thus, these studies revealed a previously uncharacterized role for InlA in facilitating systemic spread via the lymphatic system after invasion of the gut mucosa.     

Brucella lipopolysaccharide acts as a virulence factor

Brucella is a facultative intracellular bacterium responsible for brucellosis. Virulence factors involved in Brucella replication and Brucella's strategies to circumvent the immune response are under investigation. VirB proteins that form the type IV secretion system and that are involved in intracellular replication are considered as one of Brucella's virulence factors. In addition to this secretion system, bacterial outer membrane components have also been described as being implicated in Brucella survival in the host. For example, this bacterium possesses an unconventional non-endotoxic lipopolysaccharide that confers resistance to anti-microbial attacks and modulates the host immune response. These properties make lipopolysaccharide an important virulence factor for Brucella survival and replication in the host.     

The Prognostic Value of Harvested Lymph Nodes and the Metastatic Lymph Node Ratio for Gastric Cancer Patients: Results of a Study of 1,101 Patients

Aim

To investigate whether the recommendation to remove 15 lymph nodes that is used in the staging system is necessary to assess gastric cancer progression and to evaluate whether our metastatic lymph node ratio dividing method, adapted from the AJCC’s (American Joint Committee on Cancer) 7^th TNM staging system, is helpful for the patients with fewer than 15 harvested lymph nodes.

Methods

We performed a retrospective study of 1101 patients with histologically diagnosed gastric cancer who underwent a D2 gastrectomy at the Sun Yat-sen University Cancer Center between January 2001 and December 2010. The Kappa and Chi-squared tests were employed to compare the clinicopathological variables. The Kaplan-Meier method and Cox regression were employed for the univariate and multivariate survival analyses.

Results

In the trial, 346, 601 and 154 patients had 0–14, 15–30 and more than 30 lymph nodes harvested, respectively. The median survival times of patients with different lymph nodes harvested in N0, N1, N2 and N3a groups were 45.43, 54.28 and 66.95 months (p = 0.068); 49.22, 44.25 and 56.72 months (p<0.001), 43.94, 47.97 and 35.19 months (p = 0.042); 32.88, 42.76 and 23.50 months (p = 0.016). Dividing the patients who had fewer than 15 lymph nodes harvested by the metastatic lymph node ratio at 0, 0.13 and 0.40, the median survival times of these 4 groups were 70.6, 50.5, 53.5 and 30.7 months (p<0.001). After re-categorising these 4 groups into the N0, N1, N2, N3a groups, the histological grade, T staging, premier N staging, and restaged N staging were the independent prognostic factors.

Conclusions

Large numbers of lymph nodes harvested in radical gastrectomy do not cause stage migration. For those patients with a small number of harvested lymph nodes, their stage should be divided by the metastatic lymph node ratio, referred to in the TNM staging system, to assign them an accurate stage.