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.
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.
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.
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.
Development of an in vivo imaging method to assess lymphatic draining function in the K/B×N mouse model of inflammatory arthritis.
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.
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.
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.
Near infrared; lymphatic drainage; lymphangiogenesis; inflammation; lymph nodes; in vivo imagining
Investigation of the effect of lymphatic inhibition on joint and draining lymph node pathology during the course of arthritis progression in mice.
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.
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.
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.
Lymphatic drainage; lymphangiogenesis; inflammation; lymph nodes; in vivo imagining
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.
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.
mouse; lymph node; lymphatic drainage; Evans Blue; lymphography
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.
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.
B-cells; Inflammation; Rheumatoid Arthritis; Lymph nodes; B cell depletion therapy
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.
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.
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).
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.
Knee; Popliteal lymph node; Western population; Magnetic resonance imaging; Popliteal fossa
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.
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.
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.
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.
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.
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.
Breast neoplasms; Primary systemic therapy; Sentinel lymph node biopsy
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.
Breast cancer related lymphoedema; Pathophysiology; VEGF-C
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.
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.
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.
Footpad immunization; alternative; Hock immunization; CFA; IACUC regulations; immune response
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.
Salmonella typhimurium; green fluorescent protein; lymph node metastasis; red fluorescent protein
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.
Cervical, axillary, cubital, inguinal, popliteal, and mesenteric lymph nodes from subjects of various ages who had died a sudden death were examined histologically. Care was taken to establish by morphometry the proportional distribution in lymph node cross-sections of cortical, paracortical, and medullary areas. In addition, numbers and surface areas of cross-sectioned germinal centres were registered. Important differences related to age and anatomical site of lymph nodes were established by this survey. Germinal centre formation, particularly evident in infants and children, less so in young adults, and often absent in ageing individuals, was most impressive in lymph nodes normally exposed to antigenic stimulation (mesenteric and cervical lymph nodes). Paracortical and medullary areas exhibited a slight but gradual reduction with advancing age. Replacement of lymphatic parenchyma by fat tissue (lipomatous atrophy) was a characteristic of more peripheral lymph nodes usually subjected to little antigenic stimulation, that is, cubital, axillary, and popliteal nodes. It should be emphasised that both age-related and regional differences have to be taken into account in a meaningful functional interpretation of lymph node morphology.
There is a clinical need for improved intraoperative detection of lymph node metastases from malignant melanoma (MM). We aimed to investigate the use of recombinant vaccinia virus GLV-1h68, expressing green fluorescent protein (GFP), for real-time intraoperative detection of melanoma lymph node metastases in an immunocompetent animal model. Mice bearing foot pad tumors received intratumoral injections of GLV-1h68 and 48 hours later were evaluated for popliteal lymph node metastasis using noninvasive bioluminescence imaging and fluorescence imaging. Histologic analysis of lymph nodes was performed to determine sensitivity and specificity of virus-mediated detection. Intratumoral injection of GLV-1h68 into primary foot pad melanoma tumors resulted in viral transmission to popliteal lymph nodes, infection of lymphatic metastases, and transgene expression that was reliably and easily detected. Histologic confirmation demonstrated favorable operating characteristics of this assay (sensitivity 80%, specificity 100%, positive predictive value [PPV] 100%, negative predictive value [NPV] 91%). Detection of marker gene expression by GLV-1h68 allowed the detection of lymphatic metastases in an immunocompetent animal model of MM. This assay is rapid, sensitive, specific, and easy to perform and interpret. As a candidate gene therapy virus for killing cancer, GLV-1h68 may also have significant concomitant diagnostic utility in the staging of cancer patients.
Malignant melanoma; sentinel lymph node biopsy; oncolytic virus
The pressure in the cutaneous lymphatic capillaries of normal mice anesthetized with nembutal ranged between 0.0 and 2.7 cm. of water. Measurements of the interstitial pressure in the tissue immediately next the lymphatics showed that, in more than half the instances studied, there was a slight gradient of pressure from the tissues to the lymph. In nearly all the other instances the pressures inside and outside the lymphatic capillaries were approximately equal. In two cases in which lymph flow in the capillaries was rapid, the lymph pressure may have been negative. Under these circumstances there must have been a considerable gradient of pressure from the tissues to the lymph. In skin which was rapidly becoming, or had recently become, edematous as result of the application of xylol or of heat, the intralymphatic capillary pressure generally was increased, yet when compared with the pressure prevailing in the edema fluid outside of the capillaries it was usually found to be relatively much lower, at times by as much as 5.9 cm. of water. The findings indicate that a pressure gradient is an important factor in lymph formation under normal and pathological circumstances.
Heparanase expression has been linked to increased tumor invasion, metastasis, and angiogenesis and with poor prognosis. The aim of the study was to monitor the effect of heparanase expression on lymph node metastasis, in heparanase-overexpressing subcutaneous Eb mouse T-lymphoma tumors, and their draining lymph node. Dynamic contrast-enhanced magnetic resonance imaging (MRI) using biotin-BSA-GdDTPA-FAM/ROX was applied for analysis of blood volume, vascular permeability, and interstitial convection, and for detection of very early stages of such metastatic dissemination. Eb tumors increased extravasation, interstitial convection, and lymphatic drain of the contrast material. Interstitial flow directions were mapped by showing radial outflow interrupted in some tumors by directional flow toward the popliteal lymph node. Heparanase expression significantly increased contrast enhancement of the popliteal lymph node but not of the primary tumor. Changes in MR contrast enhancement preceded the formation of pathologically detectable metastases, and were detectable when only a few enhanced green fluorescent protein (EGFP)-expressing Eb cells were found near and within the nodes. These results demonstrate very early, heparanase-dependent vascular changes in lymph nodes that were visible by MRI following administration of biotin-BSA-GdDTPA-FAM/ROX, and can be used for studying the initial stages of lymph node infiltration.
Heparanase; vascular permeability; lymphatic drain; lymphangiography; MRI
The purpose of this study was to determine the effect of dendritic cell (DC) transfers on the incidence of diabetes in female nonobese diabetic (NOD) mice. Groups of 4-wk-old NOD female mice were given a single foot pad of DCs (70-90% purity) isolated from the draining lymph nodes (LN) of the pancreas (PLN), the cervical LNs, or the axillary/inguinal LNs. In addition, other groups of NOD mice received purified spleen DCs, purified PLN T cells (the major contaminating population in DC preparations), or the injection vehicle PBS. All groups were monitored for diabetes for one year. Significant protection from diabetes was observed in NOD mice receiving greater than 1 x 10(4) PLN DCs in comparison to mice receiving other DCs populations, PLN T cells, or PBS (P less than 0.05). The pancreata of NOD mice that received PLN DCs demonstrated significantly lower levels of lymphocytic infiltration in the islets that age-sex matched nondiabetic female NOD control mice (P less than 0.05). LN cells from nondiabetic NOD mice that received PLN DC protected irradiated female recipients from the adoptive transfer of diabetes to a greater degree than LN cells from age and sex matched nondiabetic female NOD mice that did not receive PLN DC transfers at 36 d (P = 0.014) and at 1 yr (P = 0.0015) after transfer. These data suggest that the PLN DC transfers are able to modulate autoimmunity and limit diabetes expression in the NOD mouse. PLN DCs transfers may regulate autoimmunity by the induction of regulatory cells.
To investigate the effectiveness of subcutaneous administration for targeting antibody to lymph nodes we have used data on the kinetics of anti-H-2Kk antibody in Kk-positive and Kk-negative mice to construct a compartmental model of antibody biodistribution. Modeling analysis revealed that antibody injected subcutaneously into negative mice leaves the injection site via convection of material in newly formed lymph, rapidly enters the nodes of the lymphatic chain and finally enters the bloodstream, presumably by the thoracic duct. A small fraction of lymph is shunted around the popliteal nodes (25%) and around the lumbar nodes (15%). The kinetics of antibody injected subcutaneously into positive mice are defined largely by the binding of antibody to nearby cells. As a result of such binding, antibody is retained in the foot and the popliteal and lumbar nodes. The modeling analysis predicts dose-dependent kinetics: increasing dose results in increased uptake in the nodes. Doses that saturate the nodes can be expected to appear in the blood, thus increasing systemic toxicity and reducing signal to noise ratio.
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.