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1.  Power Doppler Ultrasound Phenotyping of Expanding versus Collapsed Popliteal Lymph Nodes in Murine Inflammatory Arthritis 
PLoS ONE  2013;8(9):e73766.
Rheumatoid arthritis is a chronic inflammatory disease manifested by episodic flares in affected joints that are challenging to predict and treat. Longitudinal contrast enhanced-MRI (CE-MRI) of inflammatory arthritis in tumor necrosis factor-transgenic (TNF-Tg) mice has demonstrated that popliteal lymph nodes (PLN) increase in volume and contrast enhancement during the pre-arthritic “expanding” phase of the disease, and then suddenly “collapse” during knee flare. Given the potential of this biomarker of arthritic flare, we aimed to develop a more cost-effective means of phenotyping PLN using ultrasound (US) imaging. Initially we attempted to recapitulate CE-MRI of PLN with subcutaneous footpad injection of US microbubbles (DEFINITY®). While this approach allowed for phenotyping via quantification of lymphatic sinuses in PLN, which showed a dramatic decrease in collapsed PLN versus expanding or wild-type (WT) PLN, electron microscopy demonstrated that DEFINITY® injection also resulted in destruction of the lymphatic vessels afferent to the PLN. In contrast, Power Doppler (PD) US is innocuous to and efficiently quantifies blood flow within PLN of WT and TNF-Tg mice. PD-US demonstrated that expanding PLN have a significantly higher normalized PD volume (NPDV) versus collapsed PLN (0.553±0.007 vs. 0.008±0.003; p<0.05). Moreover, we define the upper (>0.030) and lower (<0.016) quartile NPDVs in this cohort of mice, which serve as conservative thresholds to phenotype PLN as expanding and collapsed, respectively. Interestingly, of the 12 PLN phenotyped by the two methods, there was disagreement in 4 cases in which they were determined to be expanding by CE-MRI and collapsed by PD-US. Since the adjacent knee had evidence of synovitis in all 4 cases, we concluded that the PD-US phenotyping was correct, and that this approach is currently the safest and most cost-effective in vivo approach to phenotype murine PLN as a biomarker of arthritic flare.
doi:10.1371/journal.pone.0073766
PMCID: PMC3767819  PMID: 24040061
2.  CD23+/CD21hi B-cell translocation and ipsilateral lymph node collapse is associated with asymmetric arthritic flare in TNF-Tg mice 
Arthritis Research & Therapy  2011;13(4):R138.
Introduction
Rheumatoid arthritis (RA) is a chronic autoimmune disease with episodic flares in affected joints. However, how arthritic flare occurs only in select joints during a systemic autoimmune disease remains an enigma. To better understand these observations, we developed longitudinal imaging outcomes of synovitis and lymphatic flow in mouse models of RA, and identified that asymmetric knee flare is associated with ipsilateral popliteal lymph node (PLN) collapse and the translocation of CD23+/CD21hi B-cells (B-in) into the paracortical sinus space of the node. In order to understand the relationship between this B-in translocation and lymph drainage from flaring joints, we tested the hypothesis that asymmetric tumor necrosis factor (TNF)-induced knee arthritis is associated with ipsilateral PLN and iliac lymph node (ILN) collapse, B-in translocation, and decreased afferent lymphatic flow.
Methods
TNF transgenic (Tg) mice with asymmetric knee arthritis were identified by contrast-enhanced (CE) magnetic resonance imaging (MRI), and PLN were phenotyped as "expanding" or "collapsed" using LNcap threshold = 30 (Arbitrary Unit (AU)). Inflammatory-erosive arthritis was confirmed by histology. Afferent lymphatic flow to PLN and ILN was quantified by near infrared imaging of injected indocyanine green (NIR-ICG). The B-in population in PLN and ILN was assessed by immunohistochemistry (IHC) and flow cytometry. Linear regression analyses of ipsilateral knee synovial volume and afferent lymphatic flow to PLN and ILN were performed.
Results
Afferent lymph flow to collapsed nodes was significantly lower (P < 0.05) than flow to expanding nodes by NIR-ICG imaging, and this occurred ipsilaterally. While both collapsed and expanding PLN and ILN had a significant increase (P < 0.05) of B-in compared to wild type (WT) and pre-arthritic TNF-Tg nodes, B-in of expanding lymph nodes (LN) resided in follicular areas while B-in of collapsed LN were present within LYVE-1+ lymphatic vessels. A significant correlation (P < 0.002) was noted in afferent lymphatic flow between ipsilateral PLN and ILN during knee synovitis.
Conclusions
Asymmetric knee arthritis in TNF-Tg mice occurs simultaneously with ipsilateral PLN and ILN collapse. This is likely due to translocation of the expanded B-in population to the lumen of the lymphatic vessels, resulting in a dramatic decrease in afferent lymphatic flow. PLN collapse phenotype can serve as a new biomarker of knee flare.
doi:10.1186/ar3452
PMCID: PMC3239381  PMID: 21884592
3.  Near infrared lymphatic imaging demonstrates the dynamics of lymph flow and lymphangiogenesis during the acute vs. chronic phases of arthritis in mice 
Arthritis and rheumatism  2010;62(7):1881-1889.
Objective
Development of an in vivo imaging method to assess lymphatic draining function in the K/B×N mouse model of inflammatory arthritis.
Methods
Indocyanine green (ICG), a near-infrared (NIR) fluorescent dye, was injected intradermally into the footpad of wild-type mice, the limb was illuminated with an 806 nm NIR laser, and the movement of ICG from the injection site to the draining popliteal lymph node (PLN) was recorded with a CCD camera. ICG-NIR images were analyzed to obtain 5 measures of lymphatic function across time. K/B×N arthritic mice and control non-arthritic littermates were imaged at one-month of age when acute joint inflammation commenced, and repeated at 3 months when joint inflammation became chronic. Lymphangiogenesis in PLNs was assessed by immunochemistry.
Results
ICG and its transport within lymphatic vessels were readily visualized and quantitative measures derived. During the acute phase of arthritis, the lymphatic vessels were dilated with increased ICG signal intensity and lymphatic pulses, and PLNs became fluorescent quickly. During the chronic phase, new lymphatic vessels were present near the foot. However, ICG appearance in lymphatic vessels was delayed. The size and area of PLN lymphatic sinuses progressively increased in the K/B×N mice.
Conclusion
ICG-NIR lymphatic imaging is a valuable method to assess the lymphatic draining function in mice with inflammatory arthritis. ICG-NIR imaging of K/B×N mice identified two distinct lymphatic phenotypes during the acute and chronic phase of inflammation. This technique can be used to assess new therapies for lymphatic disorders.
doi:10.1002/art.27464
PMCID: PMC2902657  PMID: 20309866
Near infrared; lymphatic drainage; lymphangiogenesis; inflammation; lymph nodes; in vivo imagining
4.  Inhibition of lymphangiogenesis and lymphatic drainage via VEGFR-3 blockade increases the severity of inflammation in chronic inflammatory arthritis 
Arthritis and rheumatism  2009;60(9):2666-2676.
Object
Investigation of the effect of lymphatic inhibition on joint and draining lymph node pathology during the course of arthritis progression in mice.
Method
TNF transgenic (TNF-Tg) mice were used as a model of chronic inflammatory arthritis. Mice received contrast enhanced MRI to obtain ankle and knee joint synovial volumes and draining popliteal lymph node (PLN) volumes before and 8 weeks after treatment with VEGFR-3 or VEGFR-2 neutralizing antibodies, or isotype IgG. The animals were subjected to near-infrared lymphatic imaging to determine the effect of VEGFR-3 neutralization on lymph transport from paws to draining PLNs prior to sacrifice. Lymphatic vessel formation and morphology of joints and PLNs were examined by histology, immunohistochemistry, and RT-PCR.
Results
Compared to IgG treatment, VEGFR-3 neutralizing antibody treatment significantly decreased the size of PLNs, the number of lymphatic vessels in joints and PLNs, the lymphatic drainage from paws to PLNs, and the number of VEGF-C expressing CD11b+ myeloid cells in PLNs. However, it increased the synovial volumes and inflammatory area in ankle and knee joints. VEGFR-2 neutralizing antibody, in contrast, inhibited both lymphangiogenesis and joint inflammation.
Conclusion
Lymphangiogenesis and lymphatic drainage are reciprocally related to the severity of joint lesions during the development of chronic arthritis. Lymphatic drainage plays a beneficial role in controlling the progression of chronic inflammation.
doi:10.1002/art.24764
PMCID: PMC2810533  PMID: 19714652
Lymphatic drainage; lymphangiogenesis; inflammation; lymph nodes; in vivo imagining
5.  Response in distant lymph nodes of mice to infection in the hind footpad with Mycobacterium marinum. 
Infection and Immunity  1980;28(1):225-229.
In an attempt to demonstrate the importance of the popliteal lymph node in limiting the progress of infection with Mycobacterium marinum in the hind footpads of C57BL mice, such infections were studied in mice subjected to popliteal or popliteal and inguinal adenectomies. In the absence of the popliteal node, the footpad infection was only slightly enhanced compared with infections of sham-operated control mice; the inguinal node was found to be greatly enlarged and appeared to have substituted for the absent popliteal node. In the absence of both popliteal and inguinal nodes, the disease process in the footpads was again only slightly enhanced, and the axillary node appeared to have enlarged greatly and to have functionally replaced the missing, more proximate nodes. In additional experiments, mice subjected to adenectomy only on one side and injected in that hind footpad with phytohemagglutinin or India ink demonstrated hypertrophy or deposition of carbon particles in the more distant node only on the side of the injection. Thus, there appear to be rather direct functional connections among popliteal, inguinal, and axillary nodes that do not depend on blood circulation.
Images
PMCID: PMC550916  PMID: 7380564
6.  Efficacy of B cell Depletion Therapy on Joint Flare is Associated with Increased Lymphatic Flow 
Arthritis and rheumatism  2013;65(1):130-138.
Objective
B cell depletion therapy (BCDT) ameliorates rheumatoid arthritis by mechanisms that are incompletely understood. Arthritic flare in tumor necrosis factor transgenic (TNF-Tg) mice is associated with efferent lymph node (LN) “collapse,” triggered by B cell translocation into lymphatic spaces and decreased lymphatic drainage. We examined whether BCDT efficacy is associated with restoration of lymphatic drainage due to removal of obstructing nodal B cells.
Methods
We developed contrast-enhancement (CE) MRI imaging, near-infrared indocyanine green (NIR-ICG) imaging, and intravital immunofluorescent imaging to longitudinally assess synovitis, lymphatic flow, and cell migration in lymphatic vessels in TNF-Tg mice. We tested to see if BCDT efficacy is associated with restoration of lymphatic draining and cell egress from arthritic joints.
Results
Unlike active lymphatics to normal and pre-arthritic knees, afferent lymphatic vessels to collapsed LNs in inflamed knees do not pulse. Intravital immunofluorescent imaging demonstrated that CD11b+ monocytes/macrophages in lymphatic vessels afferent to expanding LN travel at high velocity (186 ± 37 micrometer/sec), while these cells are stationary in lymphatic vessels afferent to collapsed PLN. BCDT of flaring TNF-Tg mice significantly decreased knee synovial volume by 50% from the baseline level, and significantly increased lymphatic clearance versus placebo (p<0.05). This increased lymphatic drainage restored macrophages egress from inflamed joints without recovery of the lymphatic pulse.
Conclusion
These results support a novel mechanism in which BCDT of flaring joints lessens inflammation by increasing lymphatic drainage and subsequent migration of cells and cytokines from the synovial space.
doi:10.1002/art.37709
PMCID: PMC3535508  PMID: 23002006
Rheumatoid Arthritis (RA); Flare; Tumor Necrosis Factor (TNF); B cells in Inflamed Lymph Nodes (B-in); Lymphatic Pulse
7.  Mouse Model of Lymph Node Metastasis via Afferent Lymphatic Vessels for Development of Imaging Modalities 
PLoS ONE  2013;8(2):e55797.
Animal studies of lymph node metastasis are constrained by limitations in the techniques available for noninvasive monitoring of the progression of lymph node metastasis, as well as difficulties in the establishment of appropriate animal models. To overcome these challenges, this study has developed a mouse model of inter-lymph-node metastasis via afferent lymphatic vessels for use in the development of imaging modalities. We used 14- to 18-week-old MRL/MpJ−/lpr/lpr (MRL/lpr) mice exhibiting remarkable systemic lymphadenopathy, with proper axillary lymph nodes (proper-ALNs) and subiliac lymph nodes (SiLNs) that are 6 to 12 mm in diameter (similar in size to human lymph nodes). When KM-Luc/GFP malignant fibrous histiocytoma-like cells stably expressing the firefly luciferase gene were injected into the SiLN, metastasis could be detected in the proper-ALN within 3 to 9 days, using in vivo bioluminescence imaging. The metastasis route was found to be via the efferent lymphatic vessels of the SiLN, and metastasis incidence depended on the number of cells injected, the injection duration and the SiLN volume. Three-dimensional contrast-enhanced high-frequency ultrasound imaging showed that the blood vessel volume and density in the metastasized proper-ALN significantly increased at 14 days after tumor cell inoculation into the SiLN. The present metastasis model, with lymph nodes similar in size to those of humans, has potential use in the development of ultrasound imaging with high-precision and high-sensitivity as well as other imaging modalities for the detection of blood vessels in lymph nodes during the progression of metastasis.
doi:10.1371/journal.pone.0055797
PMCID: PMC3565997  PMID: 23405215
8.  Impact of Molecular Weight on Lymphatic Drainage of a Biopolymer-Based Imaging Agent 
Pharmaceutics  2012;4(2):276-295.
New lymphatic imaging technologies are needed to better assess immune function and cancer progression and treatment. Lymphatic uptake depends mainly on particle size (10–100 nm) and charge. The size of carriers for imaging and drug delivery can be optimized to maximize lymphatic uptake, localize chemotherapy to lymphatic metastases, and enable visualization of treatment deposition. Toward this end, female BALB/c mice were injected subcutaneously in the hind footpad or forearm with a series of six different molecular weight hyaluronan (HA) near-infrared dye (HA-IR820) conjugates (ca. 5–200 nm). Mice were imaged using whole body fluorescent imaging over two weeks. HA-IR820 fluorescence was clearly visualized in the draining lymphatic capillaries, and in the popliteal and iliac or axillary lymph nodes. The 74-kDa HA-IR820 had the largest lymph node area-under-the-curve. In contrast to prior reports, mice bearing limb tumors exhibited three-fold longer retention of 74-kDa HA-IR820 in the popliteal node compared to mice without tumors. HA conjugate kinetics and disposition can be specifically tailored by altering their molecular weight. The specific lymphatic uptake and increased nodal retention of HA conjugates indicate significant potential for development as a natural biopolymer for intralymphatic drug delivery and imaging.
doi:10.3390/pharmaceutics4020276
PMCID: PMC3834911  PMID: 24300232
lymphatic imaging; hyaluronan; fluorescence; nanoparticle; tumor metastasis
9.  Lymph Node Mapping in the Mouse 
Journal of immunological methods  2007;332(1-2):170-174.
Accurate identification of lymph nodes in the mouse is critical for studies of tumor metastasis, and of regional immune responses following immunization. However, these small lymphatic organs are often difficult to identify in mice using standard dissection techniques, so that larger rats have been used to characterize rodent lymphatic drainage. We developed techniques injecting dye into the mouse footpad or tail, to label the lymphatic drainage of the hind leg and flank, pelvic viscera, prostate and mammary glands. While lymphatic drainage patterns were similar in mice and rats, the inguinal lymph nodes showed distinct differences in afferent and efferent drainage. These techniques allow accurate and rapid identification of lymph nodes and lymphatic drainage in normal as well as diseased mice.
doi:10.1016/j.jim.2007.11.012
PMCID: PMC2342937  PMID: 18164026
mouse; lymph node; lymphatic drainage; Evans Blue; lymphography
10.  Direct visualization of changes of lymphatic function and drainage pathways in lymph node metastasis of B16F10 melanoma using near-infrared fluorescence imaging 
Biomedical Optics Express  2013;4(6):967-977.
The lymphatic system provides an initial route for cancer cell dissemination in many cancers including melanoma. However, it is largely unknown how the lymphatic system changes during tumor progression due in part to the lack of imaging techniques currently available. In this study, we non-invasively imaged changes of lymphatic function and drainage patterns using near-infrared fluorescence (NIRF) imaging. Dynamic NIRF imaging following intradermal injection of indocyanine green (ICG) was conducted in C57BL/6 mice prior to inoculation of B16F10 murine melanoma cells to the dorsal aspect of the left hindpaw for baseline data or directly to the popliteal lymph node (PLN) and until 21 days post-implantation (p.i.). A series of acquired fluorescent images were quantified to measure lymphatic contractile function. Computed tomography (CT) was also performed to measure the volume of tumor-draining lymph nodes (LNs). We observed significant reduction of lymphatic contractility from 7 days p.i. until 21 days p.i.. Altered lymphatic drainage patterns were also detected at 21 days p.i. in mice with tumor in the paw and at 11 days p.i. in mice with tumor in the PLN, due to lymphatic obstruction of normal lymphatic drainages caused by extensive tumor invasion of draining LNs. Since lymphatic function and architecture were progressively altered during tumor growth and metastasis, non-invasive NIRF imaging may provide a new method to stage disease. In addition, this novel technique can be used as a diagnostic method to non-invasively assess lymphatic response as mechanism of therapeutic action.
doi:10.1364/BOE.4.000967
PMCID: PMC3675875  PMID: 23761026
(170.0170) Medical optics and biotechnology; (170.2655) Functional monitoring and imaging; (170.3880) Medical and biological imaging; (170.4580) Optical diagnostics for medicine
11.  Expanded CD23+/CD21hi B cells in inflamed lymph nodes are associated with the onset of inflammatory-erosive arthritis in TNF-transgenic mice and are targets of anti-CD20 therapy1 
Anti-CD20 B cell depletion therapy (BCDT) is very effective for some patients with rheumatoid arthritis (RA), however the pathogenic role of B lymphocytes in RA and the primary targets of BCDT are unknown. The human TNF transgenic (hTNF-tg) mouse model of RA displays a chronic-progressive disease that spreads from distal to proximal joints, and is generally considered to be adaptive immune system-independent. We have previously reported that knee arthritis in hTNF-tg mice is accompanied by structural and functional changes of the adjoining popliteal lymph node (PLN), detectable by contrast-enhanced magnetic resonance imaging (CE-MRI). To better understand these changes, here we show that onset of knee synovitis and focal erosions are paralleled by PLN contraction and accumulation of large numbers of B cells in the lymphatic sinus spaces within the node. Flow cytometry from 2, 4-5, and 8-12 month old TNF-tg mice demonstrated that B cell accumulation in the PLN follows ankle arthritis, but commences before knee disease, and involves early expansion of CD21hi, CD23+, IgMhi, CD1d+, activation marker-negative, polyclonal B cells which are found to be specifically restricted to lymph nodes draining inflamed, arthritic joints. The same B cell population also accumulates in PLNs of K/BxN mice with autoantigen-dependent arthritis. Strikingly, we show that BCDT ameliorates hTNF-tg disease and clears follicular and CD21hi, CD23+ B cells from the PLNs. Based on these findings, we propose a model whereby B cells contribute to arthritis in mice, and possibly RA, by directly affecting the structure, composition and function of joint-draining lymph nodes.
doi:10.4049/jimmunol.0903489
PMCID: PMC2874087  PMID: 20435928
B-cells; Inflammation; Rheumatoid Arthritis; Lymph nodes; B cell depletion therapy
12.  Lymphangiogenesis in Regional Lymph Nodes Is an Independent Prognostic Marker in Rectal Cancer Patients after Neoadjuvant Treatment 
PLoS ONE  2011;6(11):e27402.
One of the major prognostic factors in rectal cancer is lymph node metastasis. The formation of lymph node metastases is dependent on the existence of a premetastatic niche. An important factor preceding metastasis are lymph vessels which are located in the lymph node. Accordingly, the occurrence of intranodal lymphangiogenesis is thought to indicate distant metastasis and worse prognosis. To evaluate the significance of lymph node lymphangiogenesis, we studied formalin fixed, paraffin embedded adenocarcinomas and regional lymph nodes of 203 rectal cancer patients who were treated with neoadjuvant radiochemotherapy and consecutive curative surgery with cancer free surgical margins (R0). Regional lymph node lymph vessels were detected by immunohistochemistry for podoplanin (D2-40). Our results show that the presence of lymphatic vessels in regional lymph nodes significantly affects the disease-free survival in univariate and multivariate analyses. In contrast, there was no correlation between peritumoral or intratumoral lymph vessel density and prognosis. Indeed, our study demonstrates the importance of lymphangiogenesis in regional lymph nodes after neoadjuvant radiochemotherapy and consecutive surgery as an independent prognostic marker. Staining for intranodal lymphangiogenesis and methods of intravital imaging of lymphangiogenesis and lymphatic flow may be a useful strategy to predict long-term outcome in rectal cancer patients. Furthermore, addition of VEGF-blocking agents to standardized neoadjuvant treatment schemes might be indicated in advanced rectal cancer.
doi:10.1371/journal.pone.0027402
PMCID: PMC3210168  PMID: 22087309
13.  Features of the popliteal lymph nodes seen on musculoskeletal MRI in a Western population 
Skeletal Radiology  2011;40(8):1041-1045.
Objective
To asses the features and explore the clinical relevance of popliteal lymph nodes (PLNs) detected on MRI examination for different pathologies of the knee.
Materials and methods
A total of 150 knee MRIs, which were conducted for various indications, were retrospectively collected from the Picture Archiving and Communication System. Imaging planes in at least two orthogonal planes were mandatory, with a field of view extending 15 cm cranial from the joint space. The localization of the PLN was determined by measuring the distance of the lowest border of the PLN to the lowest border of the lateral femoral condyle. Clinical diagnosis was obtained from radiology reports and a statistician performed the statistical analysis.
Results
The patients were 70 males [mean age 36.6 years (range: 5–72 years)] and 80 females [mean age 41.1 years (range: 9–76 years)]. In 36.7% of the patients, a PLN was visible. The number of PLNs was negatively associated with age (p < 0.001). The mean number of PLNs was 0.5 PLN per patient. The mean length, height, and width were respectively: 0.57 cm (SD = 0.15), 0.84 cm (SD = 0.26), and 0.71 cm (SD = 0.23). The mean location was 5.8 cm (SD = 1.61). No association was found between the presence of PLNs and internal derangement, inflammation, or cancer (p = 0.368).
Conclusions
PLNs appearance is age related, with a higher frequency at a young age. The presence of the PLNs showed no relation to a specific clinical situation.
doi:10.1007/s00256-010-1093-z
PMCID: PMC3125512  PMID: 21249495
Knee; Popliteal lymph node; Western population; Magnetic resonance imaging; Popliteal fossa
14.  Binding of Candida albicans yeast cells to mouse popliteal lymph node tissue is mediated by macrophages. 
Infection and Immunity  1993;61(8):3244-3249.
We previously reported that Candida albicans yeast cells adhere to the macrophage-rich medullary and subcapsular sinus areas of mouse lymph node tissue. To determine whether the yeast cell-lymph node interaction is mediated by macrophages, the effect of specific elimination of macrophages on yeast cell binding was studied, and yeast cell adherence was correlated with the ingestion of India ink by lymph node cells. Macrophage elimination was done by use of liposome-containing dichloromethylene diphosphonate (L-Cl2MDP). Mice were injected in the hind footpads with the L-Cl2MDP preparation, popliteal lymph nodes were removed 5 days later, and yeast cell adherence was determined by an ex vivo binding assay. As controls, lymph nodes from mice that received footpad injections of either phosphate-buffered saline (PBS) alone or liposome-containing PBS were used. Use of macrophage- and neutrophil-specific monoclonal antibodies in tissue immunostaining showed that the L-Cl2MDP treatment eliminated macrophages but not neutrophils from the medullary and subcapsular sinus areas of the popliteal lymph nodes. A striking reduction of yeast cell adherence occurred with lymph nodes from L-Cl2MDP-treated mice compared with lymph nodes from control animals. The lymph node-yeast cell binding patterns of L-Cl2MDP-treated and control mice were the same regardless of mouse strain, sex, or T-cell competency. Results of India ink experiments, in which India ink was injected into footpads of mice and was rapidly taken up by popliteal lymph node macrophages, showed a strong correlation between yeast adherence and India ink staining of cells. In addition, the interaction of yeast cells with lymph node tissue from normal mice was not significantly affected by the addition of two extracellular matrix proteins, fibronectin and laminin, during the ex vivo adherence assay. These data indicate that medullary and subcapsular sinus lymph node macrophages express an adhesion system similar to that described for mouse splenic marginal zone macrophages.
Images
PMCID: PMC280995  PMID: 8335357
15.  Lymph-node staining with activated carbon CH40: a new method for axillary lymph-node dissection in breast cancer 
Canadian Journal of Surgery  2000;43(3):191-196.
Objective
To demonstrate the usefulness of activated carbon particles (CH40) as a vital staining dye for visualizing lymphatic vessels and lymph nodes in breast cancer.
Design
A retrospective evaluation.
Setting
Department of Surgery in Sendai National Hospital, Japan, a 716-bed teaching hospital.
Methods
To identify as many lymph nodes as possible in the axillary fat, by which we might decrease the possibility of the presence of undetected metastatic nodes, an emulsion of activated carbon particles (CH40) was injected into the centre of the mammary gland, close to the tumour site, 3 days before radical surgery.
Main outcome measure
The number of lymph nodes found by the traditional method and by the CH40-injection method were recorded.
Results
After injection, the CH40 was readily adsorbed into regional lymphatics and streamed along with the lymph flow to blacken regional lymph nodes. The CH40-guided method increased the mean number of nodes per case found in the axilla from 8.4, by the traditional method, to 14.0 nodes per case.
Conclusions
The use of the CH40 technique has two technical advantages; one is that it allows surgeons to locate the blackened lymph nodes at the time of surgery and the other is that it allows pathologists to look for the nodes in fatty tissue. Lymph-node dissection with the aid of activated carbon particles is inexpensive, easy to perform and enables the smallest lymph nodes to be easily recognized. CH40 is the technique of choice for the detection of axillary lymph nodes in cases where the number of lymph nodes detected by the traditional method is too small for accurate surgery. In conclusion, the present study demonstrates that CH40 could be an appropriate tool for more accurate staging of breast cancer axillary specimens.
PMCID: PMC3695160  PMID: 10851412
16.  Growth of a transplantable lymphoma and its modification in mice infected with the inducing virus. 
British Journal of Cancer  1976;34(4):390-400.
The growth of a transplantable lymphoma was examined in normal mice and in mice previously infected with the lymphoma-inducing virus (ULV). Normal BALB/c mice respond to a footpad injection of X-irradiated lymphoma cells (ULMC) with popliteal lymph node (PLN) enlargement; mice previously infected with ULV do not. 106 viable ULMC injected into the footpads of ULV-infected mice grew progressively, and the animals died with disseminating malignant lymphoma. In contrast, this dose of cells injected into normal animals evoked strong host responses in the foot and draining lymph node, and no progressive growth of the lymphoma occurred. This increased susceptibility of the ULV-infected animals was also observed when ULMC were injected s.c. into the back or i.m. into the calf muscle, but not after s.c. injection of an unrelated 3-methylcholanthrene-induced sarcoma. Resistance to tumour growth after i.v. injection of ULMC is clearly ineffective, since 10 cells can grow and kill the animal, and in this case no increased susceptibility of ULV-infected animals was observed.
Images
PMCID: PMC2025249  PMID: 184811
17.  An interstitial hypothesis for breast cancer related lymphoedema 
Breast cancer related lymphoedema (BCRL), the chronically swollen arm of patients that have been treated for breast cancer, is no longer considered to be a result of lymphatic obstruction as recent studies have identified failing peripheral lymphatic function as a principal contributing factor. The aetiology and pathophysiology that results in this lymphatic failure is not clearly understood, but it can occur with minimal or even in some cases no damage to the axillary lymph nodes, and evidence suggests that some patients are pre-disposed to develop the disease, and have poor lymphatic function in their non-affected arms. It has been shown that interstitial forces such as hydrostatic pressure, and interstitial fluid velocity, can regulate both lymph flow, and lymph formation, and there is good evidence that interstitial forces are dysregulated in lymphoedema patients. Here I outline a hypothesis for how dysregulation of interstitial parameters could contribute to the generation of breast cancer related lymphoedema, by combining disparate strands of current evidence on the molecular and physiological control of interstitial and lymph flows. One mechanism by which lymphoedema could be generated is that a reduction in interstitial velocity results in increased VEGF-C production, which in low flow conditions, instead of acting on the lymphatics to increase pumping and lymphangiogenesis, acts on vasculature to increase fluid filtration. The resulting increase in interstitial pressure restores flow, but at the expense of increased volume and hence oedema. The evidence supporting the hypothesis and possible tests of it are presented and discussed.
doi:10.1016/j.pathophys.2009.10.006
PMCID: PMC2923079  PMID: 19963358
Breast cancer related lymphoedema; Pathophysiology; VEGF-C
18.  Feasibility of Sentinel Lymph Node Biopsy in Breast Cancer Patients with Initial Axillary Lymph Node Metastasis after Primary Systemic Therapy 
Journal of Breast Cancer  2011;14(2):147-152.
Purpose
Primary systemic therapy (PST) downstages up to 40% of initial documented axillary lymph node (ALN) metastases in breast cancer. The current surgical treatment after PST consists of breast tumor resection and axillary lymph node dissection (ALND). This strategy, however, does not eliminate unnecessary ALND in patients with complete remission of axillary metastases. The aim of this study was to examine the accuracy of sentinel lymph node biopsy (SLNB) after PST among patients with documented ALN metastasis at presentation and to identify the rate of pathologic complete-remission (CR) with ALN after PST.
Methods
We analyzed 66 patients with ALN metastasis that was pathologically proven preoperatively who underwent SLNB and concomitant ALND after PST. Axillary ultrasound (AUS) was used to evaluate the clinical response of initially documented ALN metastasis after PST. Intraoperative lymphatic mapping was performed using blue dye with or without radioisotope.
Results
After PST, 34.8% of patients had clinical CR of ALN on AUS and 28.8% patients had pathologic CR of ALN. The overall success rate of SLNB after PST was 87.9%, and the sentinel lymph node identification rate in patients with clinical CR was 95.7%. In patients with successful lymphatic mapping, 70.7% of patients had residual axillary metastases. The overall accuracy and false-negative rate were 87.9% and 17.1% in all patients: 95.5% and 10.0% in patients with clinical CR of ALN, and 83.3% and 19.4% in patients with residual axillary disease after PST.
Conclusion
Our findings suggest that SLNB may be feasible in patients with initial documented ALN metastasis who have clinical CR for metastatic ALN after PST. Further investigation in a prospective setting should be performed to confirm our results.
doi:10.4048/jbc.2011.14.2.147
PMCID: PMC3148541  PMID: 21847411
Breast neoplasms; Primary systemic therapy; Sentinel lymph node biopsy
19.  Oral tolerance originates in the intestinal immune system and relies on antigen carriage by dendritic cells 
Oral tolerance induction is a key feature of intestinal immunity, generating systemic nonresponsiveness to ingested antigens. In this study, we report that orally applied soluble antigens are exclusively recognized in the intestinal immune system, particularly in the mesenteric lymph nodes. Consequently, the initiation of oral tolerance is impeded by mesenteric lymphadenectomy. Small bowel transplantation reveals that mesenteric lymph nodes require afferent lymph to accomplish the recognition of orally applied antigens. Finally, oral tolerance cannot be induced in CCR7-deficient mice that display impaired migration of dendritic cells from the intestine to the mesenteric lymph nodes, suggesting that immunologically relevant antigen is transported in a cell-bound fashion. These results demonstrate that antigen transport via afferent lymphatics into the draining mesenteric lymph nodes is obligatory for oral tolerance induction, inspiring new therapeutic strategies to exploit oral tolerance induction for the prevention and treatment of autoimmune diseases.
doi:10.1084/jem.20052016
PMCID: PMC2118247  PMID: 16533884
20.  MECHANISM OF GRAFT-VERSUS-HOST-INDUCED LYMPHADENOPATHY IN MICE  
The Journal of Experimental Medicine  1973;137(5):1293-1302.
Graft-vs.-host (GVH)-induced lymphadenopathy of the popliteal lymph node has been produced in C57BL/6 x A/J F1 (BAF1) mice by injecting A/J spleen cells into the rear footpads. By giving 51Cr-labeled BAF1 lymphoid cells intravenously to the hosts, 24 h before sacrifice, we have demonstrated that a large portion of the GVH-induced lymphadenopathy is due to the trapping of circuating lymphocytes in the challenged lymph nodes. Most of the remaining enlargement can be attributed to proliferation of host cells within the reacting lymph nodes. Conditions have been defined under which the weights and [14C]thymidine incorporation of the popliteal nodes can be plotted against the dose of injected A/J spleen cells on a double-log scale to give a linear dose-response. The popliteal lymph node GVH assay is a simple and effective means of quantitating immune reactivity to histocompatibility antigens in mice.
PMCID: PMC2139240  PMID: 4144986
21.  Experimental murine leprosy: growth of Mycobacterium lepraemurium in C3H and C57/BL mice after footpad inoculation. 
Infection and Immunity  1975;12(3):480-489.
Forty-three female C57/BL and C3H mice were inoculated with 2.7 X 10(6) Mycobacterium lepraemurium into each hind footpad. The foot thickness and the number of acid-fast bacilli in the footpad and popliteal and inquinal lymph nodes were recorded. In addition the morphological index and the mean bacillary length were determined in the footpad and in the popliteal lymph node. The bacilli multiplied in both strains during the first 4 weeks after inoculation. After that time no further increase in acid-fast bacilli was observed in the C57/BL strain; the bacilli became elongated and the morphological index decreased. These changes were preceded by a local swelling of the footpad due to the onset of an immune reaction. Thus, under the present conditions, C57/BL mice were able to resist experimental infection with M. lepraemurium by developing an immune response. In C3H mice no indication of an immune reaction was detected, and the bacilli continued to multiply throughout the observation period. The mouse footpad model seems to provide an excellent basis for the use of experimental murine leprosy to study immunity to mycobacterial infections. Certain aspects of the present model are discussed in relation to the mouse footpad model as used in the study of M. leprae infection in mice.
PMCID: PMC415312  PMID: 1100521
22.  Hock immunization: A humane alternative to mouse footpad injections 
Journal of immunological methods  2007;328(1-2):204-214.
Footpad injection is a commonly used immunization method in mice. Being relatively easy to do with well-characterized lymphatic drainage, it has become a very useful immunization protocol to study local immune responses in draining lymph nodes. However, its disadvantages include use of only hind feet as a routine site of immunization since mice use their fore feet for food handling, and exacerbation of inflammation and swelling at the injection site leading to unrelieved pain and distress since feet are weight-bearing structures. With increasingly stringent Institutional guidelines for animal manipulations, there is increasing need for more humane protocols. A novel immunization protocol involving injection into the hock, the lateral tarsal region just above the ankle, a non-weight bearing structure draining to the same lymph node as the footpad, retains the advantages of footpad immunization without its drawbacks. This study, comparing immune responses between footpad and hock immunization in six different inbred mouse strains to two different protein antigens and a heat-killed bacterium, shows that hock immunization is a better alternative to footpad immunization, inducing comparable immune responses and being considerably more humane.
doi:10.1016/j.jim.2007.08.004
PMCID: PMC2464360  PMID: 17804011
Footpad immunization; alternative; Hock immunization; CFA; IACUC regulations; immune response
23.  THE RÔLE OF THE LYMPHOCYTE IN ANTIBODY FORMATION 
Following the injection of typhoid antigen or sheep erythrocytes into the pad of the rabbit's hind foot, lymph from the efferent lymphatic of the popliteal lymph node was collected and analyzed for antibody content. On separating the lymphocytes from the lymph plasma, it was found that the antibody titer of the cell extract was substantially and consistently higher than that of the surrounding fluid. This difference was greatest at the time of greatest rate of increase of antibody titer in the whole lymph, rather than when the antibody titer of the lymph plasma was highest. These results can only be interpreted to mean that the lymphocytes either produce antibodies or take them up from the lymph plasma. Incubation in vitro of lymphocytes containing one species of antibody with lymph plasma containing another showed that antibodies pass from the cells to the supernatant lymph fluid to reach approximate equilibrium; acquisition of antibody from supernatant lymph fluid was not observed. Similar results were obtained when normal lymphocytes were allowed to incubate in vivo in their own lymph fluid to which antibodies had been added. It was again found that antibodies were not absorbed or adsorbed by lymphocytes. These results seem to indicate that lymphocytes are instrumental in the formation of antibodies.
PMCID: PMC2135527  PMID: 19871445
24.  CANCER METASTASIS DIRECTLY ERADICATED BY TARGETED THERAPY WITHA MODIFIED SALMONELLA TYPHYMURIUM 
Journal of cellular biochemistry  2009;106(6):992-998.
Cancer metastasis is the life-threatening aspect of cancer and is usually resistant to standard treatment. We report here a targeted therapy strategy for cancer metastasis using a modified strain of Salmonella typhimurium. The genetically modified strain of S. typhimurium is auxotrophic for the amino acids arginine and leucine. These mutations preclude growth in normal tissue but do not reduce bacterial virulence in tumor cells. The tumor-targeting strain of S. typhimurium, termed A1-R and expressing green fluorescent protein (GFP), was administered to both axillary lymph and popliteal lymph node metastasis of human pancreatic cancer and fibrosarcoma, respectively, as well as lung metastasis of the fibrosarcoma in nude mice. The bacteria were delivered via a lymphatic channel to target the lymph-node metastases and systemically via the tail vein to target the lung metastasis. The cancer cells expressed red fluorescent protein (RFP) in the cytoplasm and GFP in the nucleus linked to histone H2B, enabling color-coded real-time imaging of the bacteria targeting the metastatic tumors. After 7–21 days of treatment, the metastases were eradicated without the need of chemotherapy or any other treatment. No adverse effects were observed. This new strategy demonstrates the clinical potential of targeting and curing cancer metastasis with engineered bacteria without the need of toxic chemotherapy.
doi:10.1002/jcb.22078
PMCID: PMC2779055  PMID: 19199339
Salmonella typhimurium; green fluorescent protein; lymph node metastasis; red fluorescent protein
25.  The prognosis and possible cause of severe primary lymphoedema. 
We studied 372 patients with primary lymphoedema in order to predict the extent and severity of the disease. We found that the limits of oedema were defined early in the process and that the loss of distal lymphatics alone did not lead to severe oedema. Severe lymphoedema was associated with pelvic lymphatic 'obstruction' on lymphography and 26% of these patients eventually required surgery. Lymphography suggested that the 'obstruction' was related to lymph nodes and inguinal node biopsies were taken at the time of lymphography in 72 patients. In patients with pelvic lymphatic 'obstruction' we found a severe nodal fibrosis which was not apparent in those with distal lymphatic disease alone. This fibrosis was not related to episodes of cellulitis and since it was present in the early stages of the disease it is unlikely to be due to slow obliteration of distal lymphatics. Furthermore it could not be reproduced by ligating either afferent or efferent lymphatics of the rabbit popliteal lymph node. This suggests that severe primary lymphoedema may develop as a result of disease of the pelvic lymph nodes.
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PMCID: PMC2492713  PMID: 6742737

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