Human Sentinel Research Articles

Non‐invasive sentinel lymph node mapping and needle guidance using clinical handheld photoacoustic imaging system in small animal

Translating photoacoustic imaging (PAI) into clinical setup is a challenge. Handheld clinical real-time PAI systems are not common. In this work, we report an integrated photoacoustic (PA) and clinical ultrasound imaging system by combining light delivery with the ultrasound probe for sentinel lymph node imaging and needle guidance in small animal. The open access clinical ultrasound platform allows seamless integration of PAI resulting in the development of handheld real-time PAI probe. Both methylene blue and indocyanine green were used for mapping the sentinel lymph node using 675 and 690 nm wavelength illuminations, respectively. Additionally, needle guidance with combined ultrasound and PAI was demonstrated using this imaging system. Up to 1.5 cm imaging depth was observed with a 10 Hz laser at an imaging frame rate of 5 frames per second, which is sufficient for future translation into human sentinel lymph node imaging and needle guidance for fine needle aspiration biopsy.

γ-Tilmanocept, a New Radiopharmaceutical Tracer for Cancer Sentinel Lymph Nodes, Binds to the Mannose Receptor (CD206).

γ-Tilmanocept ((99m)Tc-labeled-tilmanocept or [(99m)Tc]-tilmanocept) is the first mannose-containing, receptor-directed, radiolabeled tracer for the highly sensitive imaging of sentinel lymph nodes in solid tumor staging. To elucidate the mannose-binding receptor that retains tilmanocept in this microenvironment, human macrophages were used that have high expression of the C-type lectin mannose receptor (MR; CD206). Cy3-labeled tilmanocept exhibited high specificity binding to macrophages that was nearly abolished in competitive inhibition experiments. Furthermore, Cy3-tilmanocept binding was markedly reduced on macrophages deficient in the MR by small interfering RNA treatment and was increased on MR-transfected HEK 293 cells. Finally, confocal microscopy revealed colocalization of Cy3-tilmanocept with the macrophage membrane MR and binding of labeled tilmanocept to MR(+) cells (macrophages and/or dendritic cells) in human sentinel lymph node tissues. Together these data provide strong evidence that CD206 is a major binding receptor for γ-tilmanocept. Identification of CD206 as the γ-tilmanocept-binding receptor enables opportunities for designing receptor-targeted advanced imaging agents and therapeutics for cancer and other diseases.

Melanoma-Derived Wnt5a Promotes Local Dendritic-Cell Expression of IDO and Immunotolerance: Opportunities for Pharmacologic Enhancement of Immunotherapy.

The β-catenin signaling pathway has been demonstrated to promote the development of a tolerogenic dendritic cell (DC) population capable of driving regulatory T-cell (Treg) differentiation. Further studies have implicated tolerogenic DCs in promoting carcinogenesis in preclinical models. The molecular mechanisms underlying the establishment of immune tolerance by this DC population are poorly understood, and the methods by which developing cancers can co-opt this pathway to subvert immune surveillance are currently unknown. This work demonstrates that melanoma-derived Wnt5a ligand upregulates the durable expression and activity of the indoleamine 2,3-dioxygenase-1 (IDO) enzyme by local DCs in a manner that depends upon the β-catenin signaling pathway. These data indicate that Wnt5a-conditioned DCs promote the differentiation of Tregs in an IDO-dependent manner, and that this process serves to suppress melanoma immune surveillance. We further show that the genetic silencing of the PORCN membrane-bound O-acyl transferase, which is necessary for melanoma Wnt ligand secretion, enhances antitumor T-cell immunity, and that the pharmacologic inhibition of this enzyme synergistically suppresses melanoma progression when combined with anti-CTLA-4 antibody therapy. Finally, our data suggest that β-catenin signaling activity, based on a target gene expression profile that includes IDO in human sentinel lymph node-derived DCs, is associated with melanoma disease burden and diminished progression-free survival. This work implicates the Wnt-β-catenin signaling pathway as a novel therapeutic target in the melanoma immune microenvironment and demonstrates the potential impact of manipulating DC function as a strategy for optimizing tumor immunotherapy.

Superparamagnetic iron oxide-enhanced interstitial magnetic resonance lymphography to detect a sentinel lymph node in tongue cancer patients

Conclusion: This is the first report on human sentinel node (SN) detection by interstitial magnetic resonance (MR) lymphography with superparamagnetic iron oxide (SPIO) in tongue cancer patients who also underwent lymphoscintigraphy. Our results indicate that further studies are warranted, as this novel method may replace current scintigraphic techniques. Objectives: To examine the feasibility of interstitial MR lymphography using SPIO for SN detection in the head and neck region. Methods: MR images were acquired sequentially at 10 min, 30 min, and 24 h after submucosally injecting 0.1 ml SPIO (ferucarbotran) around the tumor in three patients with tongue cancer without cervical lymph node metastasis (clinical T2N0M0). Results: The SNs were clearly visualized in the 10 min interstitial MR lymphography images and were completely concordant with those visualized by 99mTc-radiocolloid lymphoscintigraphy and a gamma probe in all cases. Iron incorporation into the SNs was confirmed by pathological examination.

Estimation of Population Size and Dispersal ofAedes polynesiensison Toamaromotu, French Polynesia

Mark-release-recapture methods were used to compare Aedes polynesiensis Marks adult numbers and dispersal between dry and wet seasons in a closed population on a small island (motu) in French Polynesia. Females were more than three times more common during wet (December 2008) than dry (May 2007) season samplings although high numbers of vectors were collected during both seasons. Lincoln-Petersen estimates for Ae. polynesiensis females on the motu were 6,055 per hectare for the dry season and 18,860 per hectare for the wet season. Marked females dispersed rapidly to all parts of the motu and survived until recaptures on days 1-5 after release. Males were not adequately sampled using human sentinels or Biogent Sentinel traps.

Lysophosphatidic Acid Induces Lymphangiogenesis and IL-8 Production in Vitro in Human Lymphatic Endothelial Cells

The bioactive phospholipid lysophosphatidic acid (LPA) and its receptors LPA(1-3) are aberrantly expressed in many types of human cancer. LPA has been reported to induce tumor cell proliferation, migration, and cytokine production. However, whether LPA exerts an effect on lymphatic endothelial cells (LECs) or on lymphangiogenesis, a process of new lymphatic vessel formation that is associated with increased metastasis and poor prognosis in cancer patients, has been unknown. Here, we show that LPA induces cell proliferation, survival, migration, and tube formation, and promotes lymphangiogenesis in vitro in human dermal LECs. In addition, LPA induces IL-8 expression by enhancing IL-8 promoter activity via activation of the NF-κB pathway in LECs. Using IL-8 siRNA and IL-8 neutralizing antibody, we revealed that IL-8 plays an important role in LPA-induced lymphangiogenesis in vitro. Moreover, using siRNA inhibition, we discovered that LPA-induced lymphangiogenesis in vitro and IL-8 production are mediated via the LPA(2) receptor in LECs. Finally, using human sentinel afferent lymphatic vessel explants, we demonstrated that LPA up-regulates IL-8 production in the LECs of lymphatic endothelia. These studies provide the first evidence that LPA promotes lymphangiogenesis and induces IL-8 production in LECs; we also reveal a possible new role of LPA in the promotion of tumor progression, as well as metastasis, in different cancer types.

Lipoxygenase mediates invasion of intrametastatic lymphatic vessels and propagates lymph node metastasis of human mammary carcinoma xenografts in mouse

In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas.

Initial results of imaging melanoma metastasis in resected human lymph nodes using photoacoustic computed tomography

The pathological status of the sentinel lymph node is important for accurate melanoma staging, ascertaining prognosis and planning treatment. The standard procedure involves biopsy of the node and histopathological assessment of its status. Drawbacks of this examination include a finite sampling of the node with the likelihood of missing metastases, and a significant time-lag before histopathological results are available to the surgeon. We studied the applicability of photoacoustic computed tomographic imaging as an intraoperative modality for examining the status of resected human sentinel lymph nodes. We first applied the technique to image ex vivo pig lymph nodes carrying metastases-simulating melanoma cells using multiple wavelengths. The experience gained was applied to image a suspect human lymph node. We validated the photoacoustic imaging results by comparing a reconstructed slice with a histopathological section through the node. Our results suggest that photoacoustics has the potential to develop into an intraoperative imaging method to detect melanoma metastases in sentinel lymph nodes.

Establishment and Characterization of Human Lymphatic Endothelial Cell in Breast Cancer Patients Undergoing Sentinel Lymphadenectomy

Recently remarkable progress has been made in the understanding of lymphangiogenesis due to the availability of specific biomarkers. A sentinel lymph node (SLN) biopsy has already been established as a common surgical procedure, and the clinical usefulness of an SLN biopsy has been confirmed in patients with various types of cancer. In this study, a novel method has been successfully developed for the isolation of anatomically defined lymphatic endothelial cells (LECs) from human sentinel lymphatic channels during an SLN biopsy in breast cancer patients by the use of collagenase II digestion. The isolated cells were cultured in EGM2MV media with 10% fetal bovine serum (FBS) under hypoxic conditions in an atmosphere of 5% O 2, 5% CO2 and 90% N2 at 37°C. The cultured cells exhibited a monolayer with a cobblestone appearance. Immunofluorescence analysis using selective lymphangiogenesis markers showed expression of vascular endothelial growth factor receptor 3 (VEGFR-3), prospero homeobox 1 (Prox-1) and Podoplanin. Newly established LECs showed expressions of vascular endothelial growth factor (VEGF) family members except VEGF-D and corresponding VEGF receptors by the use of conventional RT-PCR. Treatment with VEGF-C156S (500 ng/mL) apparently induced phosphorylation of the protein kinase Akt, MAPK and JNK in human isolated LECs as determined by Western blot analysis. Peak induction of Akt, MAPK and JNK occurred at 10 to 15 minutes after incubation of the isolated LECs with VEGF-C156S. The use of the MTS cell proliferation assay showed a significant increase in the growth of human lymphatic endothelial cells with VEGF-C156S treatment. The effects of VEGF-C156S (500 ng/mL) on proliferation activity was significantly with 3% FBS condition alone (MTS score: 0.54±0.0104, n=3) and a 3% FBS+VEGFC156S (MTS score: 0.572±0.0061, n=3) condition. The isolated and enzymatic digested method adopted for the culture of human LECs is simple and useful for the investigation of the cellular, molecular and genomic properties of LECs.

Biting rates and developmental substrates for biting midges (Diptera: Ceratopogonidae) in Iquitos, Peru.

Biting midges (Diptera: Ceratopogonidae) were collected at 16 periurban and rural sites around Iquitos, Peru, between 17 October 1996 and 26 May 1997. Culicoides paraensis (Goeldi), the principal vector of Oropouche virus, was the most commonly collected species (9,086 flies) with Culicoides insinuatus Wirth & Blanton second (7,229 flies). Although both species were collected at all sampling sites (linear (distance surveyed approximately 25 km), C. paraensis dominated at northern collection sites (> 90%), whereas C. insinuatus prevailed at southern collection sites (> 60%). C. paraensis were collected from human sentinels at a constant rate throughout daylight hours, at similar rates during wet and dry months, and regardless of rainfall. Larval developmental substrates for C. paraensis included decaying platano (Musa x paradisiaca L. [Musaceae]) stems, stumps, flowers, fruits, and debris beneath platano trees as well as from soil beneath a fruiting mamay (Syzygium malaccense Merr. & Perry [Myrtaceae] ) tree and organic-rich mud along a lake shoreline. C. insinuatus adults likewise emerged from decaying platano and organic-rich mud along a lake shoreline, but also from debris accumulated in the axils of aguaje (Mauritia flexuosa L. [Palmae]) fronds and decaying citrus fruit. Despite high numbers of biting adults near putative substrates, adults of neither species emerged from other decomposing plant material, soil, phytotelmata, or artificial containers. Because both species of biting midges emerged in high numbers from all parts of platano (ubiquitous in Iquitos), it will be challenging to control them through sanitation.

Human sentinels for arbovirus surveillance and regional risk classification in South Australia

To assess the use of human sentinels to monitor arbovirus activity in South Australia and to use age-specific seroprevalence data from the same sentinels to classify regions according to risk from Ross River virus (RRV). Between 1 January 1992 and 15 August 1992, 4776 serum samples were obtained from Red Cross blood donors in the State. All sera were tested for the presence of total antibody (IgA, IgG and IgM) by indirect enzyme immunoassay. To test for recent infection, positive sera were further tested for IgA and IgM specific antibody to Ross River virus with a view to initiating public health interventions if necessary. The age, sex and postcode of residence of each donor were also recorded for seroepidemiological studies. Of the 4776 sera, 2952 were tested to the end of May (the end of the arbovirus season in South Australia). There was evidence of RRV infection in 248 sera (8.4%) but none had serological markers consistent with recent infection. The arbovirus season was considered non-epidemic, and no reactive public health interventions were introduced. Analysis of age-specific seroprevalence by post-code in the full bank of 4776 sera indicated that the Riverland is endemic for RRV, the Murray Mallee and Upper South East epidemic for RRV, and the remaining regions of the State at variable risk, depending on their proximity to these regions. The ongoing use of human sentinels is a new public health surveillance system for this disease. In the first year of operation of this surveillance system, a suboptimal number of sera was collected from some areas, and sera were tested only for RRV. However, the number of sera tested could easily be increased, as could the range of arboviruses covered by the testing.

Ross River virus activity along the south coast of New South Wales.

The sera of 468 blood donors and 63 domestic animals, collected from the south coast of New South Wales, were tested for the presence of antibodies to Ross River virus. Antibodies were detected in 7% of human sera, 25% of cow sera and 65% of horse sera. Using the blood donors as 'human sentinels', seroconversions were demonstrated in two donors from the Nowra-Kiama region and from a patient in the same area; none of the three had been outside of the study area during the period of seroconversion or at the time of infection. Of the 15 seropositive horses, 6 (40%) had lived continuously since birth on the farms on which they were bled. That humans and horses were infected with Ross River and not a related alphavirus was shown by microneutralization tests against Ross River virus and the other two alpha-viruses (Getah, Sindbis) known to occur in Australia.