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Development of an ex vivo lymph node explant model for identification of novel molecules active against Leishmania major.

ABSTRACT: Leishmaniasis is a vector-borne zoonotic infection affecting people in tropical and subtropical regions of the world. Current treatments for cutaneous leishmaniasis are difficult to administer, toxic, expensive, and limited in effectiveness and availability. Here we describe the development and application of a medium-throughput screening approach to identify new drug candidates for cutaneous leishmaniasis using an ex vivo lymph node explant culture (ELEC) derived from the draining lymph nodes of Leishmania major-infected mice. The ELEC supported intracellular amastigote proliferation and contained lymph node cell populations (and their secreted products) that enabled the testing of compounds within a system that mimicked the immunopathological environment of the infected host, which is known to profoundly influence parasite replication, killing, and drug efficacy. The activity of known antileishmanial drugs in the ELEC system was similar to the activity measured in peritoneal macrophages infected in vitro with L. major. Using the ELEC system, we screened a collection of 334 compounds, some of which we had demonstrated previously to be active against L. donovani, and identified 119 hits, 85% of which were confirmed to be active by determination of the 50% effective concentration (EC50). We found 24 compounds (7%) that had an in vitro therapeutic index (IVTI; 50% cytotoxic/effective concentration [CC50]/EC50) > 100; 19 of the compounds had an EC50 below 1 ?M. According to PubChem searchs, 17 of those compounds had not previously been reported to be active against Leishmania. We expect that this novel method will help to accelerate discovery of new drug candidates for treatment of cutaneous leishmaniasis.

SUBMITTER: Peniche AG

PROVIDER: S-EPMC3910746 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Development of an ex vivo lymph node explant model for identification of novel molecules active against Leishmania major.

Peniche Alex G AG Osorio Yaneth Y Renslo Adam R AR Frantz Doug E DE Melby Peter C PC Travi Bruno L BL

Antimicrobial agents and chemotherapy 20131014 1

Leishmaniasis is a vector-borne zoonotic infection affecting people in tropical and subtropical regions of the world. Current treatments for cutaneous leishmaniasis are difficult to administer, toxic, expensive, and limited in effectiveness and availability. Here we describe the development and application of a medium-throughput screening approach to identify new drug candidates for cutaneous leishmaniasis using an ex vivo lymph node explant culture (ELEC) derived from the draining lymph nodes o ...[more]

PMID: 24126577

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Project description:Lymph nodes (LNs) are common sites of metastatic invasion in breast cancer, often preceding spread to distant organs and serving as key indicators of clinical disease progression. However, the mechanisms of cancer cell invasion into LNs are not well understood. Existing in vivo models struggle to isolate the specific impacts of the tumor-draining lymph node (TDLN) milieu on cancer cell invasion due to the co-evolving relationship between TDLNs and the upstream tumor. To address these limitations, we used live ex vivo LN tissue slices with intact chemotactic function to model cancer cell spread within a spatially organized microenvironment. After showing that BRPKp110 breast cancer cells were chemoattracted to factors secreted by naïve LN tissue in a 3D migration assay, we demonstrated that ex vivo LN slices could support cancer cell seeding, invasion, and spread. This novel approach revealed dynamic, preferential cancer cell invasion within specific anatomical regions of LNs, particularly the subcapsular sinus (SCS) and cortex, as well as chemokine-rich domains of immobilized CXCL13 and CCL1. While CXCR5 was necessary for a portion of BRPKp110 invasion into naïve LNs, disruption of CXCR5/CXCL13 signaling alone was insufficient to prevent invasion towards CXCL13-rich domains. Finally, we extended this system to pre-metastatic TDLNs, where the ex vivo model predicted a lower invasion of cancer cells. The reduced invasion was not due to diminished chemokine secretion, but it correlated with elevated intranodal IL-21. In summary, this innovative ex vivo model of cancer cell spread in live LN slices provides a platform to investigate cancer invasion within the intricate tissue microenvironment, supporting time-course analysis and parallel read-outs. We anticipate that this system will enable further research into cancer-immune interactions and allow isolation of specific factors that make TDLNs resistant to cancer cell invasion, which are challenging to dissect in vivo.

Project description:PurposeThe aim of this study was to use 7 T ex vivo magnetic resonance imaging (MRI) scans to determine the size of lymph nodes (LNs) in total mesorectal excision (TME) specimens and to increase the pathological yield of LNs with MR-guided pathology.Materials and methodsTwenty-two fixated TME specimens containing adenocarcinoma were scanned on a 7 T preclinical MRI system with a T1-weighted 3-dimensional gradient echo sequence with frequency-selective lipid excitation (repetition time/echo time, 15/3 milliseconds; resolution, 0.293 mm) and a water-excited 3-dimensional multigradient echo (repetition time, 30 milliseconds; computed echo time, 6.2 milliseconds; resolution, 0.293 mm) pulse sequence.The first series of 11 TME specimens (S1) revealed the number and size of LNs on both ex vivo MRI and histopathology. The second series of 11 TME specimens (S2) was used to perform MR-guided pathology. The number, size, and percentages of yielded LNs of S1 and S2 were compared.ResultsIn all specimens (22/22), a median number of 34 LNs (interquartile range, 26-34) was revealed on ex vivo MRI compared with 14 LNs (interquartile range, 7.5-21.5) on histopathology (P = 0.003). Mean size of all LNs did not differ between the 2 series (ex vivo MRI: 2.4 vs 2.5 mm, P = 0.267; pathology: 3.6 vs 3.5 mm, P = 0.653). The median percentages of harvested LNs compared with nodes visible on ex vivo MRI per specimen for both series were not significantly different (40% vs 43%, P = 0.718). By using a size threshold of greater than 2 mm, the percentage improved to 71% (S1) and to 78% (S2, P = 0.895). The median number of harvested LNs per specimen did not increase by performing MR-guided pathology (S1, 14 LNs; S2, 20 LNs; P = 0.532).ConclusionsEx vivo MRI visualizes more LNs than (MR-guided) pathology is able to harvest. Current pathological examination was not further improved by MR guidance. The majority of LNs or LN-like structures visible on ex vivo MRI below 2 mm in size remain unexplained, which warrants a 3-dimensional approach for pathological reconstruction of specimens.

Dataset Information

Development of an ex vivo lymph node explant model for identification of novel molecules active against Leishmania major.

Publications

Development of an ex vivo lymph node explant model for identification of novel molecules active against Leishmania major.

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