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Distinct dacryoadenitides autoadoptively transferred to rabbits by different subpopulations of lymphocytes activated ex vivo.

ABSTRACT: To test whether CD4+ T cells proliferate in mixed cell reactions with autologous lacrimal gland (LG) acinar cells and whether these cells can autoadoptively transfer disease.Purified acinar cells were gamma irradiated and cocultured with peripheral blood lymphocytes. Activated CD4+ T cells were sorted by fluorescence-activated cell sorting (FACS). Unfractionated activated peripheral blood lymphocytes (UF), CD4+-enriched and CD4+-depleted T cells from an autologous mixed cell reaction were injected into the donor rabbit's remaining LG. After 4 weeks, ocular examinations were performed, and the rabbits were euthanized; LGs were removed for histopathology, immunohistochemistry, and real-time reverse transcription-polymerase chain reaction studies.CD4 T cells increased in the autologous mixed cell reaction from 20% to 80%. Tear production decreased in the induced disease/UF (ID/UF) group and declined even more in the ID/CD4+-enriched group. Tear breakup times decreased and rose bengal staining increased in all groups. All LGs exhibited significant histopathology and increased messenger RNAs for tumor necrosis factor ?. The ID/UF group exhibited the largest increases of CD4+ and rabbit T-lymphocyte antigen-positive cells. The ID/CD4+-enriched group contained fewer infiltrating CD4 cells but more eosinophils, severely altered acinar morphology, and increased fibrosis. LG of the ID/CD4+-depleted group exhibited large increases of CD18, major histocompatibility complex II, and CD4+ cells. Messenger RNAs for interleukin 2, interleukin 4, and CD4+ increased in the ID/CD4+-enriched group compared with the CD4+-depleted group.Autoreactive CD4+ effector cells activated ex vivo and autoadoptively transferred, caused what seems to be a distinct dacryoadenitis. The CD4+-depleted cell fraction also contained pathogenic effector cells capable of inducing disease.

SUBMITTER: Thomas PB

PROVIDER: S-EPMC2945426 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Distinct dacryoadenitides autoadoptively transferred to rabbits by different subpopulations of lymphocytes activated ex vivo.

Thomas Padmaja B PB Samant Deedar M DM Wang Yanru Y Selvam Shivaram S Stevenson Douglas D Gray John D JD Schechter Joel E JE Mircheff Austin K AK Trousdale Melvin D MD

Cornea 20101001 10

<h4>Purpose</h4>To test whether CD4+ T cells proliferate in mixed cell reactions with autologous lacrimal gland (LG) acinar cells and whether these cells can autoadoptively transfer disease.<h4>Methods</h4>Purified acinar cells were gamma irradiated and cocultured with peripheral blood lymphocytes. Activated CD4+ T cells were sorted by fluorescence-activated cell sorting (FACS). Unfractionated activated peripheral blood lymphocytes (UF), CD4+-enriched and CD4+-depleted T cells from an autologous ...[more]

PMID: 20577087

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Project description:Sepsis is a life-threatening condition caused by a dysregulated host response to infection. Endothelial cells (EC) are actively involved in sepsis-associated (micro)vascular disturbances and subsequent organ dysfunction. Lipopolysaccharide (LPS), a Gram-negative bacterial product, can activate EC leading to the expression of pro-inflammatory molecules. This process is molecularly regulated by specific receptors and distinct, yet poorly understood intracellular signaling pathways. LPS-induced expression of endothelial adhesion molecules E-selectin and VCAM-1 in mice was previously shown to be organ- and microvascular-specific. Here we report that also within renal microvascular beds the endothelium expresses different extents of E-selectin and VCAM-1. This heterogeneity was recapitulated in vitro in LPS-activated human umbilical vein EC (HUVEC). Within 2 h after LPS exposure, four distinct HUVEC subpopulations were visible by flow cytometric analysis detecting E-selectin and VCAM-1 protein. These encompassed E-selectin-/VCAM-1- (-/-), E-selectin+/VCAM-1- (E-sel+), E-selectin+/VCAM-1+ (+/+), and E-selectin-/VCAM-1+ (VCAM-1+) subpopulations. The formation of subpopulations was a common response of endothelial cells to LPS challenge. Using fluorescence-activated cell sorting (FACS) we demonstrated that the +/+ subpopulation also expressed the highest levels of inflammatory cytokines and chemokines. The differences in responsiveness of EC subpopulations could not be explained by differential expression of LPS receptors TLR4 and RIG-I. Functional studies, however, demonstrated that the formation of the E-sel+ subpopulation was mainly TLR4-mediated, while the formation of the +/+ subpopulation was mediated by both TLR4 and RIG-I. Pharmacological blockade of NF-?B and p38 MAPK furthermore revealed a prominent role of their signaling cascades in E-sel+ and +/+ subpopulation formation. In contrast, the VCAM-1+ subpopulation was not controlled by any of these signaling pathways. Noteworthy is the existence of a "quiescent" subpopulation that was devoid of the two adhesion molecules and did not express cytokines or chemokines despite LPS exposure. Summarizing, our findings suggest that LPS activates different signaling mechanisms in EC that drive heterogeneous expression of EC inflammatory molecules. Further characterization of the signaling pathways involved will enhance our understanding of endothelial heterogeneous responses to sepsis related stimuli and enable the future design of effective therapeutic strategies to interfere in these processes to counteract sepsis-associated organ dysfunction.

Project description:Intratumor heterogeneity caused by genetic, phenotypic or functional differences between cancer cell subpopulations is a considerable clinical challenge. Understanding subpopulation dynamics is therefore central for both optimization of existing therapy and for development of new treatment. The aim of this study was to isolate subpopulations from a primary tumor and by comparing molecular characteristics of these subpopulations, find explanations to their differing tumorigenicity. Cell subpopulations from two patient derived in vivo models of primary breast cancer, ER+ and ER-, were identified. EpCAM+ cells from the ER+ model gave rise to tumors independently of stroma cell support. The tumorigenic fraction was further divided based on SSEA-4 and CD24 expression. Both markers were expressed in ER+ breast cancer biopsies. FAC-sorted cells based on EpCAM, SSEA-4 and CD24 expression were subsequently tested for differences in functionality by in vivo tumorigenicity assay. Three out of four subpopulations of cells were tumorigenic and showed variable ability to recapitulate the marker expression of the original tumor. Whole genome expression analysis of the sorted populations disclosed high similarity in the transcriptional profiles between the tumorigenic populations. Comparing the non-tumorigenic vs the tumorigenic populations, 44 transcripts were, however, significantly differentially expressed. A subset of these, 26 identified and named genes, highly expressed in the non-tumorigenic population, predicted longer overall survival (N?=?737, p<0.0001) and distant metastasis free survival (DMFS) (N?=?1379, p<0.0001) when performing Kaplan-Meier survival analysis using the GOBO online database. The 26 gene set correlated with longer DMFS in multiple breast cancer subgroups. Copy number profiling revealed no aberrations that could explain the observed differences in tumorigenicity. This study emphasizes the functional variability among cell populations that are otherwise genomically similar, and that the risk of breast cancer recurrence can only be eliminated if the tumorigenic abilities in multiple cancer cell subpopulations are inhibited.

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Distinct dacryoadenitides autoadoptively transferred to rabbits by different subpopulations of lymphocytes activated ex vivo.

Publications

Distinct dacryoadenitides autoadoptively transferred to rabbits by different subpopulations of lymphocytes activated ex vivo.

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