KSHV manipulates Notch signaling by DLL4 and JAG1 to alter cell cycle genes in lymphatic endothelia.

Increased expression of Notch signaling pathway components is observed in Kaposi sarcoma (KS) but the mechanism underlying the manipulation of the canonical Notch pathway by the causative agent of KS, Kaposi sarcoma herpesvirus (KSHV), has not been fully elucidated. Here, we describe the mechanism through which KSHV directly modulates the expression of the Notch ligands JAG1 and DLL4 in lymphatic endothelial cells. Expression of KSHV-encoded vFLIP induces JAG1 through an NFkappaB-dependent mechanism, while vGPCR upregulates DLL4 through a mechanism dependent on ERK. Both vFLIP and vGPCR instigate functional Notch signalling through NOTCH4. Gene expression profiling showed that JAG1- or DLL4-stimulated signaling results in the suppression of genes associated with the cell cycle in adjacent lymphatic endothelial cells, indicating a role for Notch signaling in inducing cellular quiescence in these cells. Upregulation of JAG1 and DLL4 by KSHV could therefore alter the expression of cell cycle components in neighbouring uninfected cells during latent and lytic phases of viral infection, influencing cellular quiescence and plasticity. In addition, differences in signaling potency between these ligands suggest a possible complementary role for JAG1 and DLL4 in the context of KS.

Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening.

Using animal mesentery with intravital optical microscopy is a well-established experimental model for studying blood and lymph microcirculation in vivo. Recent advances in cell biology and optical techniques provide the basis for extending this model for new applications, which should generate significantly improved experimental data. This review summarizes the achievements in this specific area, including in vivo label-free blood and lymph photothermal flow cytometry, super-sensitive fluorescence image cytometry, light scattering and speckle flow cytometry, microvessel dynamic microscopy, infrared (IR) angiography, and high-speed imaging of individual cells in fast flow. The capabilities of these techniques, using the rat mesentery model, were demonstrated in various studies; e.g., real-time quantitative detection of circulating and migrating individual blood and cancer cells, studies on vascular dynamics with a focus on lymphatics under normal conditions and under different interventions (e.g. lasers, drugs, nicotine), assessment of lymphatic disturbances from experimental lymphedema, monitoring cell traffic between blood and lymph systems, and high-speed imaging of cell transient deformability in flow. In particular, the obtained results demonstrated that individual cell transportation in living organisms depends on cell type (e.g., normal blood or leukemic cells), the cellos functional state (e.g., live, apoptotic, or necrotic), and the functional status of the organism. Possible future applications, including in vivo early diagnosis and prevention of disease, monitoring immune response and apoptosis, chemo- and radio-sensitivity tests, and drug screening, are also discussed.

Study on the mechanism of regulation on the peritoneal lymphatic stomata with Chinese herbal medicine.

AIM: To study the mechanism of Chinese herbal medicine (CHM), the prescription consists of Radix Salviae Miltiorrhizae, Radix Codonopsitis Pilosulae, Rhizoma Atractylodis Alba and Rhizoma Alismatis, Leonurus Heterophyllus Sweet,etc on the regulation of the peritoneal lymphatic stomata and the ascites drainage. METHODS: The mouse model of live fibrosis was established with the application of intragastric installations of carbon tetrachloride once every three days; scanning electron microscope and computer image processing were used to detect the area and the distributive density of the peritoneal lymphatic stomata; and the concentrations and NO in the serum were measured and analyzed in the experiment. RESULTS: Two different doses of CHM could significantly increase the area of the peritoneal lymphatic stomata, promote its distributive density and enhance the drainage of urinary ion such as sodium, potassium and chlorine. Meanwhile, the NO concentration of two different doses of CHM groups was 133.52+/-23.57 micromol/L and 137.2+/-26.79 micromol/L respectively. In comparison with the control group and model groups (48.36+/-6.83 micromol/L and 35.22+/-8.94 micromol/L, P<0.01),there existed significantly marked difference, this made it clear that Chinese herbal medicine could induce high endogenous NO concentration. The effect of Chinese herbal medicine on the peritoneal lymphatic stomata and the drainage of urinary ion was altered by adding NO donor(sodium nitropurruside,SNP) or NO synthase (NOS) inhibitor (N(G)-monomethyl-L-arginine, L-NMMA) to the peritoneal cavity. CONCLUSION: There existed correlations between high NO concentration and enlargement of the peritoneal lymphatic stomata, which result in enhanced drainage of ascites. These data supported the hypothesis that Chinese herbal medicine could regulate the peritoneal lymphatic stomata by accelerating the synthesis and release of endogenous NO.

Intestinal absorption of colostral lymphocytes in newborn lambs and their role in the development of immune status.

Two model experiments were conducted to study the intestinal absorption of colostral lymphoid cells and the role of these cells in the development of immune status in newborn lambs. In experiment I, 17 lambs of 14 Merino ewes were used. Suspensions of lymphoid cells separated from the colostrum (cell density: 5 x 10(6)/ml) and blood (3 x 10(6)/ml) were labelled with technetium (Na99mTcO4) of 37 MBq/ml radioactive concentration. In three groups of lambs, 10-ml volumes of the cell suspensions were injected directly into the duodenum after laparotomy, while in a fourth group (group Ia) the same volume was administered to the animals through an oesophageal tube. The labelled cells revealed that colostral cells of the lamb's own dam are absorbed from the gastrointestinal tract and get into the newborn lamb's lymph circulation irrespective of the route of application. In experiment II, involving 40 lambs of 40 ewes, we studied the effect of absorbed colostral lymphocytes on the development of the newborn lamb's immune status. Twenty ewes (group A) each were treated with 3 ml tetanus anatoxin twice, while the remaining animals (group B) were left uninoculated. Lambs of group A (designated A2) were separated from their dams immediately after birth, then were administered, through an oesophageal tube, 10 ml of a suspension of lymphoid cells (cell density: 5 x 10(6)/ml) separated from the maternal colostrum. Subsequently, the lambs were interchanged with lambs of nonimmunized ewes of group B (designated lambs B1), i.e. were mutually put out to nursing. At three days of age, lambs of groups A1, A2, B1 and B2 were inoculated with 3 ml tetanus anatoxin, then blood samples were taken from them 5 times in a period of 27 days for comparative examination of the humoral and cellular immune reactions. The results demonstrate that lymphoid cells from the colostrum of the lambs' own dam become absorbed into the newborn lambs' lymph circulation, remain immunologically active and may transfer, besides immunological memory, also cellular activity.

Increased lymphocyte transport by lipid absorption in rat mesenteric lymphatics.

The effect of olive oil administration on lymphocyte transport through mesenteric lymphatics was examined to see the possible involvement of nutritional absorption in lymphocyte traffic from the intestinal mucosa. After the olive oil administration to rats, remarkable increase in lymphocyte flux was observed within 2 h in lymph samples collected from rats with lymphatic fistula. The use of a high-speed microscopic video system made it possible to analyze accurately the lymphocyte transport in rapid movement that could not be detected by any of the ordinary video systems. The direct observation of mesenteric collecting lymphatics by this system showed an increment of lymphocyte transport from the intestinal mucosa by lipid absorption in 2 h. The contraction frequency of intestinal collecting lymphatics was also enhanced by olive oil administration. The densitometric analysis on video image was applied to estimate the extent of lipid absorption. The combination of a high-speed video system and the densitometric analysis revealed that the increase in lymphocyte flux occurred before lipid absorption reached its maximum and also demonstrated that the lymphocyte transport returned to control levels under the maximal absorptive condition. The results suggest that the fat absorption could be an important factor influencing the lymphocyte transport in the lymphatic system of intestine.