Evidence for the origin of Kaposi''s sarcoma from lymphatic endothelium.

Previous studies utilizing enzyme histochemistry, electron microscopy, and immunohistochemistry have failed to establish the cell of origin in Kaposi's sarcoma. The authors have rigorously tested the prevailing hypothesis that the lesion defined as Kaposi's sarcoma is derived from vascular endothelial cells. They use seven markers to characterize endothelial cells: three antigens (Factor VIII-related antigen, HLA-DR/Ia, macrophage/endothelial antigens), three enzymes (5'-nucleotidase, ATPase, alkaline phosphatase), and lectin binding (Ulex europaeus I). They applied the markers first to normal skin and lymph node, and then to biopsy specimens from 40 patients with Kaposi's sarcoma. Normal blood vessel endothelium was positive for all seven markers, but normal lymphatic endothelium was negative for all of the markers except 5'-nucleotidase and Ulex europaeus lectin. The neoplastic cells in 40 cases of Kaposi's sarcoma closely resembled those of normal lymphatic endothelium but not those of blood vessel endothelium. This suggests that Kaposi's sarcoma may originate in lymphatic endothelium.     

Experimental analysis of the origin of the wing musculature in avian embryos

Summary Interspecific grafts of somites, as well as parts of the somatic plate mesoderm, have been made between quail and chick embryos (stages 12–14 H.H.) at the level of the prospective wing bud in order to examine the relationship between somites and wing bud myogenesis. The stability of the natural quail nuclear labelling makes it possible to follow the developmental fate of grafted mesodermal cells in the host embryo. Embryos examined after subsequent incubation periods of 3–7 days show the following distribution of somatic and somitic cells within the wing bud: as soon as the three zones of different cell density within the mesoderm can be distinguished, cells of somitic origin are limited to the prospective myogenic are which is made up of a mixed population of somatic and somitic cells, whereas the prospective chondrogenic area as well as the subectodermal zone only consists of cells originated from the somatic plate mesoderm. After further incubation, single muscle blastema are present which were also seen to be a mixture of somatic and somitic cells. The cells of muscular bundles are of somitic origin, while the muscle connective tissue cells are derived from the somatic plate mesoderm. After grafting into the coelomic cavity or on the chorio-allantoic membrane, fragments of the somatic plate mesoderm previously isolated from quail embryos (stage 14 H.H.) at the level of the prospective wing bud exhibit well developed skeletal elements, but fail to differentiate any musculature. These experimental investigations support previous evidence for a somitic origin of wing bud myogenic cells. Histological and scanning electron microscopic studies of the brachial somites and the adjacent somatic plate mesoderm of chick embryo (stages 13–15 H.H.) reveal that migration of still undifferentiated somitic cells into the brachial somatic plate mesoderm begins to take place in embryos at stage 14.     

The beta-chemokine receptor D6 is expressed by lymphatic endothelium and a subset of vascular tumors

The lymphatic vessels (lymphatics) play an important role in channeling fluid and leukocytes from the tissues to the secondary lymphoid organs. In addition to driving leukocyte egress from blood, chemokines have been suggested to contribute to leukocyte recirculation via the lymphatics. Previously, we have demonstrated that binding sites for several pro-inflammatory beta-chemokines are found on the endothelial cells (ECs) of lymphatics in human dermis. Here, using the MIP-1alpha isoform MIP-1alphaP, we have extended these studies to further support the contention that the in situ chemokine binding to afferent lymphatics exhibits specificity akin to that observed in vitro with the promiscuous beta-chemokine receptor D6. We have generated monoclonal antibodies to human D6 and showed D6 immunoreactivity on the ECs lining afferent lymphatics, confirmed as such by staining serial skin sections with antibodies against podoplanin, a known lymphatic EC marker. In parallel, in situ hybridization on skin with antisense D6 probes demonstrated the expression of D6 mRNA by lymphatic ECs. D6-immunoreactive lymphatics were also abundant in mucosa and submucosa of small and large intestine and appendix, but not observed in several other organs tested. In lymph nodes, D6 immunoreactivity was present on the afferent lymphatics and also in subcapsular and medullary sinuses. Tonsilar lymphatic sinuses were also D6-positive. Peripheral blood cells and the ECs of blood vessels and high endothelial venules were consistently nonreactive with anti-D6 antibodies. Additionally, we have demonstrated that D6 immunoreactivity is detectable in some malignant vascular tumors suggesting they may be derived from, or phenotypically similar to, lymphatic ECs. This is the first demonstration of chemokine receptor expression by lymphatic ECs, and suggests that D6 may influence the chemokine-driven recirculation of leukocytes through the lymphatics and modify the putative chemokine effects on the development and growth of vascular tumors.     

Comparison of the diagnostic accuracy of lymphatic endothelium markers: Bayesian approach.

Tumor lymphatic density is evaluated by means of specific lymphatic endothelium markers, and is a potential predictor of clinically meaningful outcomes. There are many claims on the postulated superiority of some of these markers to identify lymphatics, always in the absence of quantitative data. We therefore compared the diagnostic accuracy of the antibody against podoplanin and the commercially available D2-40, employing Bayesian statistics to account for the absence of a gold standard. We used the pan-endothelial marker CD34 to identify 23,542 distinct blood and lymphatic vessels in sections from 30 formalin-fixed, paraffin-embedded archival tissue blocks of head and neck squamous cell carcinoma specimens. We stained two adjacent sections with podoplanin and D2-40 and identified the continuum of each stained vessel in the sections with a comprehensive method. Overall, 1,864 vessels were stained with both markers, 119 only with podoplanin and 391 only with D2-40. Significantly more vessels with intraluminal red blood cells were stained with D2-40 compared to podoplanin (McNemar's P<0.0001). Both antibodies had extremely high specificity (99.7% (95% credible interval (CrI): 99.5-99.9%) and 98.8% (95% CrI: 98.3-99.5%) for podoplanin and D2-40, respectively) and very high sensitivity (92.6% (95% CrI: 86.1-97.9%) and 97.3% (95% CrI: 94.9-99.2%) for podoplanin and D2-40, respectively). Inferences were qualitatively similar when we took into account in the analyses the possibility that the two tests (antibodies) may be correlated. We calculated that 96.3% (95% CrI: 94.2-98.6%) of the vessels stained with podoplanin and 88.9% (95% CrI: 83.9-95.7%) of the vessels stained with D2-40 were truly lymphatics. These numbers were in agreement with the observed number of stained vessels without intraluminal red blood cells. Our results suggest that both antibodies are excellent lymphatic endothelium markers and that there may be little reason to prefer either of them in most settings.     

Experimental analysis of blood vessel development in the avian wing bud.

Shortly after its appearance, the avian limb bud becomes populated by a rich plexus of vascular channels. Formation of this plexus occurs by angiogenesis, specifically the ingrowth of branches from the dorsal aorta or cardinal veins, and by differentiation of endogenous angioblasts within limb mesoderm. However, mesenchyme located immediately beneath the surface ectoderm of the limb is devoid of patent blood vessels. The objective of this research is to ascertain whether peripheral limb mesoderm lacks angioblasts at all stages or becomes avascular secondarily during limb development. Grafts of core or peripheral wing mesoderm, identified by the presence or absence of patent channels following systemic infusion with ink, were grafted from quail embryos at stages 16-26 into the head region of chick embryos at stages 9-10. Hosts were fixed 3-5 days later and sections treated with antibodies that recognize quail endothelial cells and their precursors. Labeled endothelial cells were found intercalated into normal craniofacial blood vessels both nearby and distant from the site of implantation following grafting of limb core mesoderm from any stage. Identical results were obtained following grafting of limb peripheral mesoderm at stages 16-21. However, peripheral mesoderm from donors older than stage 22 did not contain endothelial precursors. Thus at the onset of appendicular development angioblasts are present throughout the mesoderm of the limb bud. During the fourth day of incubation, these cells are lost from peripheral mesoderm, either through emigration or degeneration.     

The orientation of the wing mesenchyme influences the direction of the migration of myoblasts in the avian embryonic wing bud

Summary In order to analyze the influence of the orientation of the wing bud mesenchyme on the proximodistal direction of the migration of myoblasts in the avian embryonic wing bud, blocks of wing-bud mesenchyme were cut out and rotated around a dorso-palmar axis through 90° or 180°. Tissues originating from the quail wing bud and containing myoblasts were grafted into the space between the wing mesenchyme and the rotated blocks of mesenchyme proximal to the latter. In all experiments the donor-embryos were older than the acceptor-embryos. From HH stage 24 on, the rotation of the block of mesenchyme inhibited the migration of the myoblasts in a distal direction. We therefore propose that the orientation of the wing bud mesenchyme has an influence on the migratory behavior of myoblasts. This influence could provide “directional information” for the migrating myoblasts, allowing the migration of myoblasts in a distal direction only.     

The expression of E-selectin and chemokines in the cultured human lymphatic endothelium with lipopolysaccharides

This study investigated the expression of selectins and chemokines in cultured human lymphatic endothelial cells stimulated with lipopolysaccharides. In microarray, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 gene expressions in the lymphatic endothelium with lipopolysaccharides did not change at 0.5 h but increased two- to three-fold at 12 h, whereas E-selectin increased 10-fold at 0.5 h and 68-fold at 12 h compared with untreated cells. The E-selectin mRNA and protein increased in the lymphatic endothelial cells with lipopolysaccharides at more than two-fold levels compared with human umbilical vein endothelial cells. Induction of Cys-Cys chemokine ligand 2, 3, 5, 7, 8 and 20 mRNAs in the lymphatic endothelial cells with lipopolysaccharides was detected in microarray and real-time PCR. The Cys-Cys chemokine ligand 2, 5 and 20 mRNA amounts in cells with high concentration lipopolysaccharides were larger in the lymphatic endothelial cells than in human umbilical vein endothelial cells. The Cys-Cys chemokine ligand 3 and 8 mRNAs were not detected in human umbilical vein endothelial cells. Induction of Cys-X-Cys chemokine ligand 1, 3, 5, 6 and 8 mRNAs was detected in the lymphatic endothelial cells with lipopolysaccharides. The Cys-X-Cys chemokine ligand 3, 5 and 8 mRNA amounts in cells with high concentration lipopolysaccharides were larger in the lymphatic endothelial cells than in human umbilical vein endothelial cells. In conclusion, it was demonstrated that the cultured human lymphatic endothelial cells express E-selectin and phagocyte-attractive chemokine genes.     

Endogenous origin of the lymphatics in the avian chorioallantoic membrane.

The lymphatics of the intestinal organs have important functions in transporting chyle toward the jugulosubclavian junction, but the lymphangiogenic potential of the splanchnic mesoderm has not yet been tested. Therefore, we studied the allantoic bud of chick and quail embryos. It is made up of endoderm and splanchnic mesoderm and fuses with the chorion to form the chorioallantoic membrane (CAM) containing both blood vessels and lymphatics. In day 3 embryos (stage 18 of Hamburger and Hamilton [HH]), the allantoic mesoderm consists of mesenchymal cells that form blood islands during stage 19 (HH). The endothelial network of the allantoic bud, some intraluminal and some mesenchymal cells express the hemangiopoietic marker QH1. The QH1-positive endothelial cells also express the vascular endothelial growth factor receptor-3 (VEGFR-3), whereas the integrating angioblasts and the round hematopoietic cells are QH1-positive/VEGFR-3-negative. The ligand, VEGF-C, is expressed ubiquitously in the allantoic bud, and later predominantly in the allantoic epithelium and the wall of larger blood vessels. Allantoic buds of stage 17-18 (HH) quail embryos were grafted homotopically into chick embryos and reincubated until day 13. In the chimeric CAMs, quail endothelial cells are present in blood vessels and lymphatics, the latter being QH1 and VEGFR-3 double-positive. QH1-positive hematopoietic cells are found at many extra- and intraembryonic sites, whereas endothelial cells are confined to the grafting site. Our results show that the early allantoic bud contains hemangioblasts and lymphangioblasts. The latter can be identified with Prox1 antibodies and mRNA probes in the allantoic mesoderm of day 4 embryos (stage 21 HH). Prox1 is a specific marker of the lymphatic endothelium throughout CAM development.     

On the origin of avian air sacs

For many vertebrates the lung is the largest and lightest organ in the body cavity and for these reasons can greatly affect an organism's shape, density, and its distribution of mass; characters that are important to locomotion. In this paper non-respiratory functions of the lung are considered along with data on the respiratory capacities and gas exchange abilities of birds and crocodilians to infer the evolutionary history of the respiratory systems of dinosaurs, including birds. From a quadrupedal ancestry theropod dinosaurs evolved a bipedal posture. Bipedalism is an impressive balancing act, especially for tall animals with massive heads. During this transition selection for good balance and agility may have helped shape pulmonary morphology. Respiratory adaptations arising for bipedalism are suggested to include a reduction in costal ventilation and the use of cuirassal ventilation with a caudad expansion of the lung into the dorsal abdominal cavity. The evolution of volant animals from bipeds required yet again a major reorganization in body form. With this transition avian air sacs may have been favored because they enhanced balance and agility in flight. Finally, I propose that these hypotheses can be tested by examining the importance of the air sacs to balance and agility in extant animals and that these data will enhance our understanding of the evolution of the respiratory system in archosaurs.     

The origin of 7 S RNA in virions of avian sarcoma viruses.

Duck and chick embryo fibroblasts were labeled with ³²PO₄^3? and cellular 7S RNA was purified. Ribonuclease T₁ digests were prepared and the resulting oligonucleotides were fractionated by two-dimensional electrophoresis. Duck 7 S RNA lacked one large oligonucleotide present in chicken 7 S RNA, thus establishing that the two species contain 7 S RNAs of different sequence. The Prague C strain of Rous sarcoma virus was grown in each type of host and the 7 S RNA found in progeny virus was examined. It was found to be identical to that of the host which produced the virus.     

Experimental analysis of blood vessel development in the avian wing bud

Shortly after its appearance, the avian limb bud becomes populated by a rich plexus of vascular channels. Formation of this plexus occurs by angiogenesis, specifically the ingrowth of branches from the dorsal aorta or cardinal veins, and by differentiation of endogenous angioblasts within limb mesoderm. However, mesenchyme located immediately beneath the surface ectoderm of the limb is devoid of patent blood vessels. The objective of this research is to ascertain whether peripheral limb mesoderm lacks angioblasts at all stages or becomes avascular secondarily during limb development. Grafts of core or peripheral wing mesoderm, identified by the presence or absence of patent channels following systemic infusion with ink, were grafted from quail embryos at stages 16–26 into the head region of chick embryos at stage 9–10. Hosts were fixed 3–5 days later and sections treated with antibodies that recognize quail endothelial cells and their precursors. Labeled endothelial cells were found intercalated into normal craniofacial blood vessels both nearby and distant from the site of implantation following grafting of limb core mesoderm from any stage. Identical results were obtained following grafting of limb peripheral mesoderm at stages 16–21. However, peripheral mesoderm from donors older than stage 22 did not contain endothelial precursors. Thus at the onset of appendicular development angioblasts are present throughout the mesoderm of the limb bud. During the fourth day of incubation, these cells are lost from peripheral mesoderm, either through emigration or degeneration.     

On the migration of epidermal melanoblasts in the avian embryonic wing bud

Summary After heterotopic grafting of quail neural crest cells to the wing buds of embryos of an unpigmented chicken strain, epidermal melanocytes of donor origin are found almost exclusively distal from the graft in the host's epidermis. This directed cell migration ceases, if the apical ectodermal ridge (together with a small amount of subridge mesoderm) is removed from the operated wing buds or if impermeable materials are interposed between it and the rest of the wing bud. Under these conditions epidermal melanocytes are found not only distal from but also proximal to the grafts. From this it may be deduced that the apical ectodermal ridge directs the migration of epidermal melanoblasts in the avian embryonic wing bud, possibly by a chemotactic mechanism. The presence or absence of the apical ectodermal ridge had no observable effect on the migratory behaviour of other neural crest derived cell populations (Schwann cells and non-epidermal melanocytes) in the wing bud. This shows that the apical ectodermal ridge specifically influences epidermal melanocytes.This work was supported by the Österreichischer Fonds zur Förderung der wissenschaftlichen Forschung (P 4680)     

Cells of origin of avian postsynaptic dorsal column pathways.

Whereas in the cervical spinal cord of pigeons lamina IV and medial lamina V neurons are at the origin of postsynaptic pathways to the dorsal column nuclei, lumbar lamina IV neurons do not project substantially beyond the cervical enlargement. There is, however a distinct group of medially located lumbar lamina V neurons which projects ipsilaterally to the dorsal column nuclei.     

On the determination of mesodermal tissues in the avian embryonic wing bud

Summary The quail-chick-marker technique has been employed to elucidate the determination of embryonic tissues in the avian wing bud. It was found that myogenic cells of somitic origin are determinated to give rise only to muscular tissue from HH-stage 19 on. Mesenchyme in the cartilage forming regions is determinated to form cartilage from HH-stage 20 on. Tissue from non-cartilage forming regions retains the option to form cartilage at least up to HH-stage 26. Whereas cells of somatopleural lineage do not migrate within the limb bud, cells of somitic origin do so up to at least HH-stage 28.This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Ch 44/4)     

Developmental origin of avian Merkel cells

We have investigated the developmental origin and ultrastructure of avian Merkel cells by electron microscopy and chick/quail transplantation experiments. On embryonic day 3, chick leg primordia were homotopically grafted onto Japanese quail host embryo. Fourteen days later, quail cells that had migrated into grafted chick legs were identified according to the masses of heterochromatin associated with the nucleolus that are characteristic for quail. Both in chick and quail, Merkel cells are usually located in the dermis just below the epidermis. They are placed between nerve terminals either individually or in small groups wrapped in sheaths that are formed by glial cell processes. Occasionally, some Merkel cells appear in nerve fascicles and within Herbst corpuscles. Merkel cells, as well as glial cells, in grafted chicken legs were of quail origin. This finding provides evidence against the epidermal origin of avian Merkel cells and indicates that Merkel cells are derived from neural crest cells that colonise, together with glial cells and melanocytes, the developing limb primordium. Accepted: 30 May 2000     

淋巴管内皮细胞透明质酸受体-1在发育小鼠肾内的表达

目的:探讨淋巴管内皮细胞透明质酸受体-1(LYVE-1)在发育小鼠肾内的表达.方法:胚胎期第13～18天(E13～18)的胎鼠和出生后第4、 14和21天(P4～21)及成年小鼠的肾,行LYVE-1免疫组织化学显色.结果:LYVE-1免疫阳性淋巴管丛最早在胚胎期第14天的小鼠肾检测到,主要位于肾皮质的动脉周围.在胚胎期第15天的肾小球中最早检测到LYVE-1免疫阳性细胞,但LYVE-1免疫阳性细胞在肾小球中的数量和位置不定.结论:LYVE-1免疫阳性细胞主要位于肾动脉周围淋巴管,在肾小球中也有LYVE-1表达.