Алгоритм ведения больных с гнойными тубовариальными образованиями

ТАЗОВЫХ ОРГАНОВ ВОСПАЛИТЕЛЬНЫЕ БОЛЕЗНИАЛГОРИТМЫНАГНОЕНИЕБОЛЬНОГО ВЕДЕНИЕ ОПТИМАЛЬНО

Lymphatic vessels: new targets for the treatment of inflammatory diseases

The lymphatic system plays an important role in the physiological control of the tissue fluid balance and in the initiation of immune responses. Recent studies have shown that lymphangiogenesis, the growth of new lymphatic vessels and/or the expansion of existing lymphatic vessels, is a characteristic feature of acute inflammatory reactions and of chronic inflammatory diseases. In these conditions, lymphatic vessel expansion occurs at the tissue level but also within the draining lymph nodes. Surprisingly, activation of lymphatic vessel function by delivery of vascular endothelial growth factor-C exerts anti-inflammatory effects in several models of cutaneous and joint inflammation. These effects are likely mediated by enhanced drainage of extravasated fluid and inflammatory cells, but also by lymphatic vessel-mediated modulation of immune responses. Although some of the underlying mechanisms are just beginning to be identified, lymphatic vessels have emerged as important targets for the development of new therapeutic strategies to treat inflammatory conditions. In this context, it is of great interest that some of the currently used anti-inflammatory drugs also potently activate lymphatic vessels

A Transgenic Prox1-Cre-tdTomato Reporter Mouse for Lymphatic Vessel Research

The lymphatic vascular system plays an active role in immune cell trafficking, inflammation and cancer spread. In order to provide an in vivo tool to improve our understanding of lymphatic vessel function in physiological and pathological conditions, we generated and characterized a tdTomato reporter mouse and crossed it with a mouse line expressing Cre recombinase under the control of the lymphatic specific promoter Prox1 in an inducible fashion. We found that the tdTomato fluorescent signal recapitulates the expression pattern of Prox1 in lymphatic vessels and other known Prox1-expressing organs. Importantly, tdTomato co-localized with the lymphatic markers Prox1, LYVE-1 and podoplanin as assessed by whole-mount immunofluorescence and FACS analysis. The tdTomato reporter was brighter than a previously established red fluorescent reporter line. We confirmed the applicability of this animal model to intravital microscopy of dendritic cell migration into and within lymphatic vessels, and to fluorescence-activated single cell analysis of lymphatic endothelial cells. Additionally, we were able to describe the early morphological changes of the lymphatic vasculature upon induction of skin inflammation. The Prox1-Cre-tdTomato reporter mouse thus shows great potential for lymphatic research

TdTomato is expressed in the skin upon crossing of the reporter mice with a K5-Cre-ERT2 line.

<p><b>A</b> Schematic representation of the breeding to K5-Cre-ERT2 and of 4-hydroxytamoxifen (4-OHT) applications on the shaved back skin (1 mg in ethanol for five consecutive days). <b>B</b> Prior (day 0) and after 4-OHT application (day 7), mice where imaged with the IVIS spectrum (excitation: 570 nm; emission: 620nm; exposure time: 10 sec; binning: HR4. Color scale min = 2600 counts, max = 13000 counts). TdTomato was expressed in the skin of double positive, treated animals. Dashed line indicates the 4-OHT treated shaved back skin.</p

The Prox1-Cre-tdTomato mouse facilitates FACS sorting of LECs.

<p>An ear single cell suspension was analyzed with a BD Fortessa flow cytometer. The tdTomato+ population contained 60% of CD45- CD31+ podoplanin+ LEC and 40% of CD45- CD31- cells. The sorting of tdTomato+ CD31+ cells allowed the isolation of a pure LEC population. Data are representative of 4 transgenic animals analyzed from 2 independent litters.</p

Comparison of the Prox1-Cre-tdTomato mouse with the Prox1-Cre-tdRFP mouse.

<p><b>A</b> Intravital confocal microscopy revealed brighter lymphatic vessels in the Prox1-Cre-tdTomato mouse than in the Prox1-Cre-tdRFP mouse (images acquired applying the same laser power and settings). <b>B</b> LVs can be visualized in Prox1-Cre-tdRFP mice only by applying a higher laser power. <b>C</b> Intravital multiphoton microscopy allowed the imaging of deep lymphatic collectors (arrowheads indicate valves), which were better visualized in Prox1-Cre-tdTomato mice. <b>D</b> Orthogonal analysis showed better visualization of the lymphatic vessel lumen in Prox1-Cre-tdTomato mice than in Prox1-mOrange mice, suggesting diffuse dye distribution in LECs. Scale bars: 100 μm.</p

The Prox1-Cre-tdTomato mouse is a valid tool for intravital microscopy of DC migration.

<p><b>A</b> Maximum intensity projection of a z-stack of a mouse ear imaged with the IVM settings. The tdTomato positive LVs were clearly visible. Scale bar: 70 μm. <b>B</b> YFP-DCs were injected into the ear and imaged for 1 hour in an inverted confocal microscope. The image corresponds to the maximum intensity projection of single frame z-stack where the migratory tracks of DCs are shown. Scale bar: 70 μm. <b>C</b> Orthogonal view of the entry of a DC into the LV is visualized over time. Four representative time points from a 1-hour video are shown. Scale bar: 20 μm.</p

TdTomato expression co-localizes with lymphatic markers in different organs.

<p><b>A</b> Whole mount preparations of ear samples showed co-localization of the lymphatic marker LYVE-1 with tdTomato (detected with an anti-RFP antibody). Dashed line indicates a lymphatic collector vessel (LYVE-1 low, tdTomato high). <b>B</b> Whole mount preparations of ear samples showed co-localization of the lymphatic marker Prox1 with tdTomato. Upper panels show lymphatic collectors, middle panels show lymphatic capillaries and lower panels show a magnification of the area indicated in the middle panels. <b>C</b> FACS single cell analyses revealed tdTomato expression in the LEC population (CD45- CD31+ podoplanin+), but not in the BEC (CD45- CD31+ podoplanin-) or in the leukocyte (CD45+) populations. <b>D</b> Ultramicroscopic analysis of an immunostained and optically cleared lymph node showed colocalization of LYVE-1 and tdTomato. Maximum projection of a 260 μm z-stack. <b>E</b> Whole mount preparation of a lymph node sample showed the presence of LYVE-1+RFP+ lymphatic endothelium in the subcapsular sinus. LYVE-1+RFP- single cells are most likely macrophages. Data are representative of 4 transgenic animals analyzed from 2 independent litters. Scale bars: 100 μm.</p

Generation of the tdTomato reporter mouse.

<p><b>A</b> Schematic representation of the LSL-tdTomato construct used for pronuclear microinjection. <b>B</b> HEK293 cells were transiently transfected with the construct with or without the STOP cassette. TdTomato fluorescence was analyzed 48 hrs post transfection with an inverted microscope and was visible only upon excision of the STOP cassette. <b>C</b> PCR genotyping of genomic DNA extracted from ear biopsies identified founder animals (f.l. founder line) positive for the transgene (tdTomato; 191 bp amplicon). A control gene (IL-2; 350 bp amplicon) was used for genomic DNA quality. <b>D</b> Transgene relative copy number was estimated by real-time PCR of genomic DNA. Primers specific for the transgene and for a control gene were used and the delta Ct was calculated. Founder 2 carried the least amount of copies, founder 4 the highest and founder 26 an intermediate number.</p