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#20583874   2010/06/29 Save this To Up

Lymphatic vessel hypertrophy in inflamed human tonsils.

The structural and molecular properties of the human tonsil lymphatic microvascular system are important to understand as these features likely contribute to fluid balance, immunity, and tumor metastasis. The tonsil is a unique lymphoid organ in that it is in intimate contact with the contents of the upper aerodigestive tract and that there are no identifiable afferent lymphatics. Conventional immunofluorescence microscopy demonstrated a remarkable degree of lymphatic vessel architecture within the tonsil; LYVE-1-positive lymphatic vessels were detected around each germinal center and in the marginal regions between the follicles. High resolution confocal laser scanning immunofluoresence microscopy demonstrated that individual lymphatic endothelial cells had a classic 'oak leaf' shape and discontinuous expression of CD31 and VE-cadherin; characteristics hypothesized to be related to fluid and cellular transport. A comparative analysis demonstrated a dramatic increase in lymphatic but not blood vessel density and complexity in inflamed compared to noninflamed tonsil tissue. The results of this study describe the spatial organization of the tonsil lymphatic vasculature, discontinuous expression of CD31 and VE-cadherin in human lymphatic capillaries, and a change in lymphatic vessel morphology in response to inflammation.

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#16261591   2006/01/31 Save this To Up

Isolation and characterization of lymphatic microvascular endothelial cells from human tonsils.

Human lymphatic endothelial cells (LECs) have isolated prevalently from human derma and tumors. As specialized lymphatic organs within the oropharynx, palatine tonsils are easily obtained and rich in lymphatic venules. Using a two-step purification method based on the sorting of endothelial cells with Ulex Europaeus Agglutinin 1 (UEA-1)-coated beads, followed by purification with monoclonal antibody D2-40, we successfully purified LECs from human palatine tonsils. The LECs were expanded on flasks coated with collagen type 1 and fibronectin for up to 8-10 passages and then analyzed for phenotypic and functional properties. Cultured cells retained the phenotypic pattern of the lymphatic endothelium of palatine tonsils and expressed functional VEGFR-3 molecules. In fact, stimulation with VEGFR-3 ligand, the vascular endothelium grow factor C, induced a marked increase in cell proliferation. Similarly to blood endothelial cells (BECs), LECs were able to form tube-like structure when seeded in Cultrex basement membrane extract. Comparative studies performed on LECs derived from palatine tonsils and iliac lymphatic vessels (ILVs), obtained with the same procedures, showed substantial discrepancies in the expression of various lymphatic markers. This points to the existence of micro- and macrovessel-derived LECs with different phenotypes, possibly involving different biological activities and functions. Palatine tonsil- and ILV-derived LECs may, therefore, represent new models for investigating function and biochemical properties of these lymphatic endothelia.

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#16257423   2006/02/03 Save this To Up

Genetic immunization with LYVE-1 cDNA yields function-blocking antibodies against native protein.

LYVE-1 is a surface bound hyaluronic acid (HA) receptor that is preferentially expressed by lymphatic endothelial cells (LEC). cDNA encoding full-length human LYVE-1 was coated onto gold particles that were then delivered via helium-assisted jet propulsion (gene gun) into the skin of Balb/C mice. LYVE-1 antisera, but not control pre-immune sera, recognized LYVE-1-transfected 293T cells by flow cytometry. While 40-70% of cultured human dermal microvascular endothelial cells (HMEC) were positive for LYVE-1 staining, human lung microvascular endothelial cells (LMEC) were negative. LYVE-1 antisera was used to effectively separate HMEC into LYVE-1 (hi) and LYVE-1(lo) populations that were enriched or depleted, respectively, for podoplanin, another LEC marker. By immunohistochemistry, LYVE-1 antisera detected CD31(lo) podoplanin(hi) lymphatic channels in normal and psoriatic human skin as well as in human tonsil. LYVE-1 antisera also blocked binding of FITC-labeled HA to HMEC (but not LMEC), demonstrating that these antibodies recognized regions of LYVE-1 required for HA binding. In summary, gene gun-assisted delivery of cDNA encoding LYVE-1 into skin resulted in reliable production of antisera that specifically and functionally recognized native LYVE-1 protein.

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#12819012   2003/06/23 Save this To Up

Molecular characterization of NF-HEV, a nuclear factor preferentially expressed in human high endothelial venules.

Lymphocyte homing to secondary lymphoid tissue and lesions of chronic inflammation is directed by multi-step interactions between the circulating cells and the specialized endothelium of high endothelial venules (HEVs). In this study, we used the PCR-based method of suppression subtractive hybridization (SSH) to identify novel HEV genes by comparing freshly purified HEV endothelial cells (HEVECs) with nasal polyp-derived microvascular endothelial cells (PMECs). By this approach, we cloned the first nuclear factor preferentially expressed in HEVECs, designated nuclear factor from HEVs (NF-HEV). Virtual Northern and Western blot analyses showed strong expression of NF-HEV in HEVECs, compared to human umbilical vein endothelial cells (HUVECs) and PMECs. In situ hybridization and immunohistochemistry revealed that NF-HEV mRNA and protein are expressed at high levels and rather selectively by HEVECs in human tonsils, Peyers's patches, and lymph nodes. The NF-HEV protein was found to contain a bipartite nuclear localization signal, and was targeted to the nucleus when ectopically expressed in HUVECs and HeLa cells. Furthermore, endogenous NF-HEV was found in situ to be confined to the nucleus of tonsillar HEVECs. Finally, threading and molecular modeling studies suggested that the amino-terminal part of NF-HEV (aa 1-60) corresponds to a novel homeodomain-like Helix-Turn-Helix (HTH) DNA-binding domain. Similarly to the atypical homeodomain transcription factor Prox-1, which plays a critical role in the induction of the lymphatic endothelium phenotype, NF-HEV may be one of the key nuclear factors that controls the specialized HEV phenotype.

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#11426486   2001/06/27 Save this To Up

Role of transforming growth factor beta1 in microvascular endothelial cell apoptosis associated with thrombotic thrombocytopenic purpura and hemolytic-uremic syndrome.

Primary human microvascular endothelial cells (MVEC) of restricted lineage undergo apoptosis when exposed to plasma from patients with thrombotic thrombocytopenic purpura (TTP) and sporadic hemolytic-uremic syndrome (HUS). This reflects the pathology and tissue distribution of lesions in vivo. As extracellular matrix (ECM) is critical to MVEC survival, and cytokines which regulate ECM, such as transforming growth factor (TGF)-beta1, have been reported anecdotally to be altered in TTP/HUS, we examined the role of TGF-beta1 and two ECM proteins, fibronectin and thrombospondin (TSP), in these disorders. Levels of active TGF-beta1 were elevated in acute but not convalescent phases of TTP/sporadic HUS, as well as TTP associated with human immunodeficiency virus infection and use of the anti-platelet drug ticlopidine. MVEC from tissues susceptible to TTP-mediated apoptosis showed little active TGF-beta1 production when exposed to TTP plasmas. In contrast, pulmonary MVEC and large-vessel EC, which are resistant to TTP-linked pathology, showed marked induction of TGF-beta1 following TTP plasma exposure. Exogenous TGF-beta1 suppressed TTP plasma-mediated apoptosis in susceptible MVEC in association with blockade of cell entry into S phase. Soluble TSP, devoid of detectable bound TGF-beta1, had a similar effect, which paralleled its ability to induce TGF-beta1 production in MVEC. In vivo, TSP deposition was diminished markedly in involved tissues of TTP patients. These data highlight the role of TGF-beta1 and ECM in TTP and suggest that differential production of TGF-beta1 by MVEC may play a role in their sensitivity or resistance to TTP/sporadic HUS-mediated apoptosis in vitro and in vivo.

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#7539445   1995/07/05 Save this To Up

Immunohistochemical characterization of human endometrial microvascular basement membrane components during the normal menstrual cycle.

The expression of three basement membrane components [collagen IV (CIV), laminin and heparan sulphate proteoglycan (HSPG)] and platelet endothelial cell adhesion molecule (PECAM) were examined by immunohistochemistry in cryostat sections of normal human endometrium. Alkaline phosphatase (ALP) was detected using enzyme histochemistry. Endometrial biopsies from the menstrual (n = 4), mid-late proliferative (n = 5), early-mid secretory (n = 5) and late secretory (n = 5) stages were collected from women with a normal menstrual cycle. At all four stages of the menstrual cycle, CIV, laminin and HSPG were expressed on basement membranes of both vessels and glands whilst PECAM expression was localized specifically to endothelial cells. A similar number of vessels/mm2 stained for CIV and laminin, as well as for PECAM at each stage of the menstrual cycle, demonstrating that all vessels in endometrium stain for these two basement membrane components. By contrast, the number of vessels/mm2 that stained positively for HSPG and ALP was significantly lower, averaging approximately 55% of the total that stained positively for PECAM, CIV and laminin. During the menstrual stage, HSPG staining intensity remained strong in glandular basement membranes but decreased dramatically in vascular basement membranes. ALP activity was variable in both the vessels and glands throughout the four stages of the menstrual cycle studied. This study demonstrates heterogeneity in basement membrane components within the endometrial microvasculature. It is postulated that the disappearance of HSPG from vascular basement membranes may play a role in the process of vascular remodelling during the menstrual stage of the cycle.

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#1730089   1992/02/18 Save this To Up

A human monoclonal autoantibody to platelet glycoprotein IIb derived from normal human lymphocytes.

Tonsillar lymphocytes from an otherwise healthy nonthrombocytopenic male child were fused with the lymphoblastoid cell line GM 4672. Twenty of 472 (4%) hybridomas had antiplatelet reactivity detected using intact platelets in an enzyme-linked immunosorbent assay. One hybridoma (STO 171) reacted to platelet glycoprotein IIb (integrin alpha IIb) as determined by radioimmunoprecipitation and immunoblotting. Antibody specificity was confirmed using immunodepletion experiments with isotypic antibodies derived from a mutlitransfused Glanzmann's thrombasthenic patient. The antibody reactivity was restricted to platelets and did not react with other integrin alpha-chain proteins expressed on granulocytes or cultured human brain-derived microvascular endothelial cells. These studies indicate that lymphocytes of normal, nonthrombocytopenic individuals have the genetic potential to produce antiplatelet autoantibodies.

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#2631918   1990/05/24 Save this To Up

Microvascular organization of human palatine tonsils.

We describe the three-dimensional organization of the microvasculature of human palatine tonsils as revealed by the vascular corrosion casting/scanning electron microscope method and light microscopy of sections. The tonsillar arteries travel in the connective tissue septa and give off many branches. They further branch into arterioles which in turn enter the follicle and the interfollicular region. These arterioles, giving off capillaries en route, reach the subepithelial region where they break up into sinusoidal capillaries. The subepithelial capillary network overlying the follicle protrudes hemispherically towards the crypt, while that overlying the interfollicular region has many switch-back loops of capillaries projected towards the crypt. The subepithelial sinusoids gather into the high endothelial venules (HEVs) which, collecting capillaries in the follicle and the interfollicular region en route, course down into the interfollicular region alongside the follicle. The HEVs surround the lateral and basal surfaces of the follicle and ultimately lead into the ordinary veins in the septa. The subepithelial sinusoids seem to be involved in taking up immunoglobulins secreted by plasma cells and any other substances released by lymphocytes and/or macrophages as well as supplying the tissues with necessary oxygen and nutrients. That the HEVs are downstream to the subepithelial sinusoids suggests that some substances which are taken up into the sinusoids and transported to the postcapillary venules induce differentiation of HEVs and maintain them.

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