Anlage of the initial part of the thoracic duct in human embryogenesis]

Canalization of the jugular lymphatic sac is accompanied with disengagement from the blood stream of the thoracic subcardinal and the thoracic parts of the superior mesocardinal veins, forming the paired thoracic anlage of the thoracic duct (ThD). Canalization of the retroperitoneal lymphatic sac (RLS) is combined with differentiation of the antevertebral plexus of the abdominal veins, formation of the ThD cistern. Lateroaortal, retrocaval and retroaortal lumbar trunks (LT) become its roots. Together with RLS and the ThD cistern they form the lymphatic "muff" around the abdominal aorta and inferior vena cava. The degree of its parts separation and its substitution by collaterals of LT and by lymph nodes determines the variants of the definitive organization of the ThD initial part. Already at the stage of anlage the intestinal trunk is not included in the ThD root system, but serves as the RLS anterior tributary, or its lumbar, preaortic tributary.     

Retroperitoneal Ancient Schwannoma: A Case Report

Schwannomas are extremely rare tumors that are composed of Schwann cells. Retroperitoneal localization comprises 0.7% to 2.6% of all schwannomas. Patients usually present with nonspecific symptoms. There are no pathognomonic features on radiologic evaluation. Preoperative biopsy is not recommended because of complication risks; however, surgery is necessary for diagnosis and treatment. Although most schwannomas are benign tumors, those that are associated with von Recklinghausen disease are malignant. Schwannomas exhibit regions of high and low cellularity, termed Antoni A and Antoni B areas, with a diffuse positivity of S100 protein on pathologic evaluation. If there are degenerative changes, such as cyst formation, hemorrhage, calcification, and hyalinization, these tumors are termed ancient schwannomas. We present a case of retroperitoneal ancient schwannoma.Key words: Retroperitoneal schwannoma, Neurilemmoma, Schwannoma, Retroperitoneal tumor, RetroperitoneumA schwannoma is a tumor of the Schwann cell or nerve cell sheath.¹ This tumor may occur anywhere there is a nerve with Schwann cells— predominantly in the head and neck region, or flexor surfaces of the extremities. Most schwannomas are benign tumors²; however, malignant schwannomas may be associated with von Recklinghausen disease.³ Retroperitoneal schwannomas are usually larger than those in other sites, and have a greater tendency to undergo spontaneous degeneration and hemorrhage when compared with head, neck, and extremity locations.⁴ We present a case of a retroperitoneal schwannoma that was reported to be an ancient schwannoma on pathologic evaluation.     

Pure choriocarcinoma of the testis presenting with jaundice: a case report and review of the literature

ABSTRACT: INTRODUCTION: Testicular cancer is the most common malignancy in men 15- to 35-years-old. The North American standard classification divides testicular cancers into germ cell tumors and non-germ cell tumors. The lymphatic spread of germ cell tumors usually involves the retroperitoneal lymph nodes. However, this spread to the retroperitoneum rarely involves the hepatic hilum. We describe an unusual case of metastatic choriocarcinoma of the testis that was clinically mimicked by a cholestatic jaundice. This is an unusual presentation of testicular cancer and, to the best of our knowledge, the first report of this kind in the literature. CASE PRESENTATION: A 28-year-old Moroccan man presented with a four-week history of progressive obstructive jaundice, and weight loss to our emergency department. Abdominal ultrasound showed a dilatation of the biliary ducts due to pathologically enlarged lymph nodes of the hepatic hilum. A complete clinical and radiologic assessment to discover the primary tumor was negative except for pulmonary metastasis. In the laboratory findings at admission there were signs of cholestasis with an abnormal increase in the rate of testicular tumor markers (serum beta-human chorionic gonadotropin level was 11,000IU/ml), which subsequently led to the suspicion of a testicular tumor. Further evaluation included testicular palpation and ultrasound which revealed a testicular nodule. The patient underwent an inguinal orchidectomy of the right testis and histopathological examination confirmed a pure choriocarcinoma. The prognosis was poor due to lymph node involvement at the hepatic hilum. He died one month later, despite general chemotherapy. CONCLUSIONS: The clinical presentation of the disease and the rarity of this entity are two remarkable characteristics described in this case report which are rarely reported in literature.     

131I-MIBG therapy in the treatment of pheochromocytoma in children--own experiences

Three cases of pheochromocytoma in children/adolescents or young adults treated by 131I-MIBG are presented in this study. In one patient 131I-MIBG was administrated after ineffective surgical treatment and chemotherapy of a benign retroperitoneal tumor, whereas in two other patients 131I-MIBG therapy was carried out because of malignant pheochromocytoma dissemination. In a child with retroperitoneal paraganglioma decrease of tumor size and its fibrosis after 131I-MIBG therapy allowed radical surgery and complete recovery. In two other cases partial remission was achieved. All patients showed a good subjective response with improvement of the general condition and better blood pressure control. In two children adverse reactions such as leucopenia, hypothyroidism or hypogonadism were observed. The presented data confirm effectiveness and acceptable tolerance of 131I-MIBG treatment in pheochromocytoma, what is very important in pediatric patients.     

The thin latissimus dorsi perforator-based free flap for resurfacing

The authors present their experience with "thin" latissimus dorsi perforator-based free flaps for resurfacing defects. Perforator-based free flaps have been used for various kinds of reconstruction by presenting important donor structures. The thin latissimus dorsi perforatorbased free flap included only the skin and superficial adipose layer to reduce its bulkiness by dissection through the superficial fascial plane. This flap was used in 12 clinical cases, without flap necrosis or other serious postoperative complications. All of the patients were examined by preoperative power Doppler ultrasound in the spectral Doppler mode to search for the most reliable perforator. This noninvasive ultrasound technique determines the exact location and course of and ensures the reliable flow of the perforators; therefore, it greatly assists microsurgeons in saving operation time and in selecting the most suitable design for perforator flap reconstruction. We used perforators that were identified several centimeters from the lateral border of the latissimus dorsi muscle. The thin flap dimensions could be safely designed for flaps measuring up to 20 cm in length and 8 cm in width for primary closure of the donor site. Generally, a long pedicle is not required for resurfacing reconstructions, where small recipient arteries in the bed are acceptable for anastomosis with pedicles. However, pedicle dissection to the proximal vessels through the latissimus dorsi muscle was required when it was necessary to match the recipient vein for anastomosis. The authors conclude that this thin latissimus dorsi perforator-based free flap has great potential for resurfacing because of its constant thickness, easy elevation with the help of power Doppler ultrasound information, and proper flap size for moderate defects caused by scar contracture release, superficial tumor ablation, and so on.     

Lower auricular malformations: their representation, correction, and embryologic correlation

Seventy-seven lower auricular malformations in 74 patients treated during the last 6 years were analyzed. Sixty cases (77.9 percent) were of malformations involving the earlobe; 54 cases involved the earlobe alone, and 6 cases were of complex deformities involving the earlobe and adjacent helix and/or tragus. Cleft earlobe was the most common lower auricular malformation (49 cases, 63.6 percent); four subtypes and their corrective methods are described. Cases of complex earlobe malformations, corrected by fabricated costal cartilage and expanded skin flap, are presented. A question mark ear (5 cases, 6.5 percent), a malformation with an ectopic anthelical fold (5 cases, 6.5 percent), and a malformation with a lower conchal stria (5 cases, 6.5 percent) are considered to be major lower auricular malformations. An attempt has been made to correlate the presented malformations with the embryologic-fetal development of the auricle. It is suggested that "clefting" ear malformations such as the cleft earlobe, the question mark ear, and the ectopic anthelical fold deformity may provide clues to understanding the embryologic-fetal development of the human auricle. It appears that hillocks 1 and 6 produce the earlobe and that hillock 4 or 5 produces the anthelix or helix.     

The use of subcutaneous pedicle flaps in the treatment of postburn scar contractures

Subcutaneous pedicle flaps, which were usually applied to repair small skin defects in the face or the fingertip, have been used with success in the treatment of 17 postburn scar contractures, with the exception of one partial flap necrosis. The results indicate the reliability and usefulness of this technique in the treatment of scar contractures, even in the extremities or the trunk. Subcutaneous pedicle flaps are effective for relatively wide contractures or quadratic contractures. When the skin tension across the contracture line is too great to use any local flap, such as a Z-plasty or V-Y plasty, the subcutaneous pedicle flap is particularly useful, because it can be freely designed in an area where the tension is small. When the flap contains some superficial scarring, the subcutaneous pedicle flap is preferred over other local flaps because of the superior vascularity and mobility.     

Underwater observations of the rare deep-sea fish <Emphasis Type="Italic">Triodon macropterus</Emphasis> (Actinopterygii,Tetraodontiformes, Triodontidae), with comments on the fine structure of the scales

Underwater observations of two living individuals and an examination of a freshly dead individual of the rare deep-sea fish Triodon macropterus revealed that they usually have the large ventral flap completely uplifted and seamlessly retracted into the abdominal region of the trunk: one individual was collected at a depth of 280 m by hook and line east of Tonaki-jima Island, Ryukyu Islands, and kept in a tank at the Okinawa Churaumi Aquarium for six years; the second living individual was observed by the ROV Hakuyo 2000 at a depth of 275 m east of Ishigaki-jima Island, Ryukyu Islands; and another individual collected at depths of 250?350 m by hook and line off Funchu Point, Yoron-jima Island (27°00.15’N, 128°26.06’E), Ryukyu Islands, provided an opportunity to demonstrate that the ventral flap could be manually uplifted by rotating the pelvis. When the ventral flap was uplifted in all of these specimens, whether naturally or manually, there were no scaleless linear bands or streaks and gaps evident in the skin between the retracted ventral flap and the abdominal region. The fine structure of the scales of the body and the ventral flap was observed by light microscopy, scanning electron microscopy, and X-ray CT scanning. The scales of the ventral flap are arranged in rows radiating from the antero-dorsal part of the ventral flap where the anterior part of the pelvis articulates with and rotates around the pectoral girdles. The orientation of the rows on the ventral flap changes from a posterior direction dorsally to a posteroventral direction more ventrally. A ridge with many sharp serrations is raised and bent from the edge of each scale on the body and ventral flap. The raised ridges on the lower scales on the ventral flap become packed together with the upper scales to form a seamless surface when the ventral flap is uplifted into the abdominal region.     

Juxta-adrenal Ancient Schwannoma: A Rare Retroperitoneal Tumor

Retroperitoneal schwannoma is a rare tumor that is often misdiagnosed as malignancy due to a concerning appearance on cross-sectional imaging. Pathology and immunohistochemistry form the gold standard for diagnosis; as such, local excision is the treatment of choice for this disease. We present two cases of juxta-adrenal ancient schwannoma that were treated with adrenalectomy and discuss the current literature regarding this entity.Key words: Ancient schwannoma, Adrenal tumor, AdrenalectomySchwannomas are tumors of the nerve sheath cells that surround peripheral nerves throughout the body. These tumors are typically benign in nature, although malignant transformation has been documented in rare cases. Schwannomas are most often found in women between the ages of 20 and 50 years; although they can be found throughout the peripheral nervous system, they seldom present in the retroperitoneum.¹ Less than 0.2% of incidental adrenal and periadrenal masses are eventually diagnosed as schwannoma,² and only 0.7% of schwannomas are found in the retroperitoneal space.^3,4 These tumors are classically well encapsulated, hypervascular, and can appear heterogeneous, making them difficult to distinguish from more concerning lesions through imaging studies alone.Schwannomas, or neurilemmomas, originate from neural crest cells and are histologically composed of spindle-shaped tumor cells organized in the Antoni A and Antoni B regions (cellularly dense and sparse, respectively).⁵ The term ancient schwannoma was coined in 1951 by Ackerman and Taylor to describe an uncommon histologic subtype with only occasional tumor cells surrounded by a hyalinized matrix, more characteristic of the degeneration associated with a long-standing and slow-growing mass.⁶Given the paucity of data regarding juxta-adrenal and other retroperitoneal ancient schwannomas, and their similar appearance to more invasive cancers, we discuss two cases of juxta-adrenal ancient schwannoma that initially presented as a malignant-appearing adrenal mass.     

Oromandibular reconstruction with the radial-forearm osteocutaneous flap: experience with 60 consecutive cases

One of the more difficult problems in reconstructive surgery of the head and neck is replacement of bone and soft tissue lost because of injury, osteomyelitis, or malignancy. The radial-forearm osteocutaneous flap is an accepted choice for oromandibular reconstruction. This study was undertaken to review one center's experience with 60 consecutive cases of oromandibular reconstruction with the radial-forearm osteocutaneous flap. Records of the 38 men and 22 women (mean age, 60 years; range, 26 to 86 years) were reviewed for tumor location, defect and bone length, flap failure rate, recipient- and donor-site complications, length of surgery, and hospital stay. Cancer resection was the reason for 97 percent of reconstructions; 33 percent of flaps were used to reconstruct a lateral defect of the mandible, 40 percent a lateral-central defect, and 27 percent a lateral-central-lateral defect. Mean skin flap size was 55 cm2 (range, 15 to 117 cm2) and mean bone length, 9.4 cm (range, 5 to 14 cm). The microvascular success rate was 98.3 percent. Complications included fracture of the donor radius (15 percent), nonunion of the mandible (5 percent), and hematoma (8.3 percent). These results are comparable to results reported in the literature with other radial forearm flaps. The free radial osteocutaneous flap is a safe and reliable choice for mandibular reconstruction. It offers sufficient bone to reconstruct large defects and can provide adequate pedicle length for vessel anastomosis to the contralateral side of the neck. The above attributes make the radial forearm osteocutaneous flap one of the "first line" flap choices for oromandibular reconstruction.     

Fine needle aspiration diagnosis of lymphangiomyomatosis. A case report

BACKGROUND: Lymphangiomyomatosis is a rare disease of females, usually of reproductive age. There is a proliferation of lymphatic smooth muscle in mediastinal, retroperitoneal and often pulmonary lymphatics and lymph nodes. CASE: A 45-year-old female presented with a right pleural effusion and increasing retroperitoneal adenopathy with palpable left inguinal adenopathy. Three months previously she had undergone a right salpingo-oophrectomy for an ovarian fibroma with concomitant left ovarian wedge biopsy, myomectomy for leiomyomas and partial omentectomy. Three years previously, at age 42, she had experienced two transient episodes of chylous pleural effusion with no sequelae. She underwent computed tomography-guided fine needle aspiration of a 4-cm inguinal lymph node to rule out lymphoma. CONCLUSION: Fine needle aspiration of lymphangiomyomatosis yields distinctive cytologic morphology. This characteristic morphology, in combination with the appropriate history, permits a minimally invasive, timely and in this particular case, entirely unexpected diagnosis.     

Severe ectropion: repair with a modified Tripier flap

Classical techniques, such as wedge resection, are well suited for mild cases of lower lid ectropion, but they often fail to cure severe cases. The reason these techniques often fail is because they address only the laxity and elongation of the ectropic lid, not the root cause. In nearly every case of severe ectropion, the root cause is importance of the pretarsal orbicularis muscle; i.e., there is inadequate muscle support for the pretarsal lower eyelid. A bipedicled musculocutaneous flap, transferred from the upper lid, was used to treat nine cases of severe lower eyelid ectropion. Eight patients had a good/excellent result. Four patients had electromyographic studies in the late postoperative period, without a single instance of even mild denervation. In cases of severe ectropion, this flap is an effective replacement for the missing skin and impotent muscle. It uses the often discarded blepharoplasty tissue, which has a perfect color and texture match. A single anatomic unit is rebuilt, transferring a strong new muscle strap with ideal supporting vectors and leaving scars in natural creases. This "blepharoplasty flap" may prove useful in other types of eyelid reconstruction.     

Thoracic duct lymphatic fluid harbors phenotypically naive T cells for use in adoptive T-cell therapy

Background aims: Manufacturing of potent chimeric antigen receptor (CAR) T cells requires phenotypically naive and early memory T cells. We hypothesized lymphatic fluid collected from the thoracic duct of children would serve as a unique reservoir for early T cells, which could then be used for CAR T-cell therapy. Methods: We evaluated lymphatic fluid collected from 25 pediatric patients undergoing thoracic duct cannulation for other clinical indications. Results: Lymphatic fluid in the thoracic duct was rich in T cells, with higher percentage of naive and stem central memory T-cell subsets compared with paired blood samples. T cells from lymphatic fluid showed decreased negative checkpoint regulators on the surface and increased rapid expansion with bead activation. Creation of CD19-directed CAR T cells from blood and lymphatic T cells showed similar lentiviral transduction properties, but CAR T cells generated from lymphatic fluid produced superior cytotoxicity in a murine leukemia model because they were able to achieve equivalent tumor eradication at lower doses. Conclusions: These results are the first characterization of T cells from the thoracic duct of pediatric patients and suggest an alternative approach for manufacturing of cellular therapy that will improve both expansion and cytotoxic effect.     

Innervated island flaps in morphofunctional vulvar reconstruction

In this article, the authors present their own experience in vulvar reconstruction following vulvectomy using two different innervated island flaps according to the size and site of the defect. Island-flap mobilization is possible thanks to the rich blood supply of the perineal region. The methods described are a "V-Y amplified sliding flap from the pubis" and a "fasciocutaneous island flap" raised from one or both gluteal folds. The V-Y amplified sliding flap from the pubis is indicated when the defect is symmetric and located anteriorly. This flap is harvested from the pubis and vascularized by the deep arterial network of the pubis. Sensory innervation is provided by branches of the ileo-inguinal nerve. The fasciocutaneous island flap, raised from one or both gluteal folds, can be used following hemivulvectomy or radical vulvectomy, respectively, to cover posteriorly located defects. Vascularization is provided by the musculocutaneous perforating branches of the pudendal artery, whereas sensory innervation is maintained through the perineal branches of the pudendal nerve. Twenty-two patients have undergone reconstructive surgery of the vulvar region from 1989 to date. On 14 patients, a V-Y amplified sliding flap was used; on 7 patients, reconstruction was carried out by island flaps raised from the gluteal fold. Both techniques are compatible with inguino-femoral lymphadenectomy, and they allow for a correct morphofunctional reconstruction and provide good local sensibility. The final result is aesthetically satisfactory, as all final scars are hidden in natural folds.     

Lymphangion coordination minimally affects mean flow in lymphatic vessels

The lymphatic system returns interstitial fluid to the central venous circulation, in part, by the cyclical contraction of a series of "lymphangion pumps" in a lymphatic vessel. The dynamics of individual lymphangions have been well characterized in vitro; their frequencies and strengths of contraction are sensitive to both preload and afterload. However, lymphangion interaction within a lymphatic vessel has been poorly characterized because it is difficult to experimentally alter properties of individual lymphangions and because the afterload of one lymphangion is coupled to the preload of another. To determine the effects of lymphangion interaction on lymph flow, we adapted an existing mathematical model of a lymphangion (characterizing lymphangion contractility, lymph viscosity, and inertia) to create a new lymphatic vessel model consisting of several lymphangions in series. The lymphatic vessel model was validated with focused experiments on bovine mesenteric lymphatic vessels in vitro. The model was then used to predict changes in lymph flow with different time delays between onset of contraction of adjacent lymphangions (coordinated case) and with different relative lymphangion contraction frequencies (noncoordinated case). Coordination of contraction had little impact on mean flow. Furthermore, orthograde and retrograde propagations of contractile waves had similar effects on flow. Model results explain why neither retrograde propagation of contractile waves nor the lack of electrical continuity between lymphangions adversely impacts flow. Because lymphangion coordination minimally affects mean flow in lymphatic vessels, lymphangions have flexibility to independently adapt to local conditions.     

Comparison and definition of spleen and lymph node: a phylogenetic analysis

The hitherto largely unsolved problem with a biological definition of spleen versus lymph node seems possible to solve from a phylogenetic point of view. Thus, it is suggested that the spleen be defined as a hemopoietic organ which is able to filter blood with sinusoids. In contradistinction, a lymph node is defined as a hemopoietic organ which is able to filter lymph with sinusoids. Comparative anatomical studies show that the spleen appears as a condensation of the lymphomyeloid complex in the spiral fold of the gut in cyclostomes. The spiral fold spleen vanishes with the bony fishes, while in cartilaginous fishes a similar spleen appears in the dorsal mesentery. The dorsal spleen remains in a retroperitoneal position in higher vertebrates and is regarded as a specialized blood vessel compartment closely connected with the blood stream. In "higher" vertebrates the spleen is a stagnated organ because splenic functions are gradually transferred to other sites. The bone marrow takes over the erythro-, thrombo- and granulocytopoiesis while the lymph nodes take over the lymphocytopoiesis. This transfer of the splenic functions is first seen in anurans and seems to be a marvelous adaptation to life on land where the need for local defence against a large number of antigens is necessary before spread of the antigens to central parts of the body. In higher vertebrates, the great number of lymph nodes at peripheral positions, derived from the lymphatic vessels, are able to do so. It is demonstrated that the definitions of spleen and lymph nodes as hemopoietic organs which by their sinusoids are able to filter blood and lymph, respectively, are not only of semantic interest but also useful in regard the immunohematological system as an entity.     

Periostin directly and indirectly promotes tumor lymphangiogenesis of head and neck cancer

Background

Metastasis to regional lymph nodes via lymphatic vessels plays a key role in cancer progression. Tumor lymphangiogenesis is known to promote lymphatic metastasis, and vascular endothelial growth factor C (VEGF-C) is a critical activator of tumor lymphangiogenesis during the process of metastasis. We previously identified periostin as an invasion- and angiogenesis-promoting factor in head and neck squamous cell carcinoma (HNSCC). In this study, we discovered a novel role for periostin in tumor lymphangiogenesis.

Methods and Findings

Periostin overexpression upregulated VEGF-C mRNA expression in HNSCC cells. By using conditioned media from periostin-overexpressing HNSCC cells, we examined tube formation of lymphatic endothelial cells. Conditioned media from periostin-overexpressing cells promoted tube formation. To know the correlation between periostin and VEGF-C, we compared Periostin expression with VEGF-C expression in 54 HNSCC cases by immunohistochemistry. Periostin expression was correlated well with VEGF-C expression in HNSCC cases. Moreover, correlation between periostin and VEGF-C secretion was observed in serum from HNSCC patients. Interestingly, periostin itself promoted tube formation of lymphatic endothelial cells independently of VEGF-C. Periostin-promoted lymphangiogenesis was mediated by Src and Akt activity. Indeed possible correlation between periostin and lymphatic status in periostin-overexpressing xenograft tumors and HNSCC cases was observed.

Conclusions

Our findings suggest that periostin itself as well as periostin-induced upregulation of VEGF-C may promote lymphangiogenesis. We suggest that periostin may be a marker for prediction of malignant behaviors in HNSCC and a potential target for future therapeutic intervention to obstruct tumoral lymphatic invasion and lymphangiogenesis in HNSCC patients.     

Periorbital lymphatic malformation: clinical course and management in 42 patients

Lymphatic malformation in the orbital cavity and surrounding region often causes disfigurement and visual problems. To better clarify the evolution and treatment of this condition, the authors studied a retrospective cohort of 42 consecutive patients seen between 1971 and 2003 and analyzed anatomic features, complications, and management. The ratio of female to male patients was 1:1. Most periorbital lymphatic malformations were noted at birth (59 percent), presenting as either unilateral swelling (60 percent) or a periorbital mass (24 percent). Sixty-two percent of lesions were on the left side. The ipsilateral cheek, temple, and forehead also were involved in 57 percent of patients. Twenty-two percent of lesions were intraconal, 30 percent were extraconal, and 48 percent were in both spaces. Forty-five percent of children had an associated cerebral developmental venous anomaly. Periorbital lymphatic malformation caused major morbidity; 52 percent of patients had intralesional bleeding and 26 percent of patients had a history of infection. Other common complications included intermittent swelling (76 percent), blepharoptosis (52 percent), proptosis (45 percent), pain (21 percent), amblyopia (33 percent), chemosis (19 percent), astigmatism (17 percent), and strabismus (7 percent). Ultimately, 40 percent of children had diminished vision and 7 percent became blind in the affected eye. Management of periorbital lymphatic malformation involved an interdisciplinary team that included an interventional radiologist, a craniofacial surgeon, and an ophthalmologist. The two therapeutic strategies were sclerotherapy (40 percent) and resection (57 percent); most patients required several interventions. A coronal approach was used for subtotal excision of fronto-temporal-orbital lymphatic malformation in 13 patients, whereas a tarsal incision was used for lesions isolated to the eyelid (n = 14). Ocular proptosis was temporarily managed by tarsorrhaphy (n = 9), but expansion of the bony orbit was needed to correct persistent proptosis (n = 8). Orbital exenteration was necessary in two patients.     

Intravital two-photon microscopy of immune cell dynamics in corneal lymphatic vessels

Background

The role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years.

Methodology/Principal Findings

We now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization.Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM). Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology.Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1–5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces.

Conclusions

To our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible physiological conditions.     

A tumor-homing peptide with a targeting specificity related to lymphatic vessels

Blood vessels of tumors carry specific markers that are usually angiogenesis-related. We previously used phage-displayed peptide libraries in vivo to identify peptides that home to tumors through the circulation and that specifically bind to the endothelia of tumor blood vessels. Here we devised a phage screening procedure that would favor tumor-homing to targets that are accessible to circulating phage, but are not blood vessels. Screening on MDA-MB-435 breast carcinoma xenografts yielded multiple copies of a phage that displays a cyclic 9-amino-acid peptide, LyP-1. Homing and binding to tumor-derived cell suspensions indicated that LyP-1 also recognizes an osteosarcoma xenograft, and spontaneous prostate and breast cancers in transgenic mice, but not two other tumor xenografts. Fluorescein-labeled LyP-1 peptide was detected in tumor structures that were positive for three lymphatic endothelial markers and negative for three blood vessel markers. LyP-1 accumulated in the nuclei of the putative lymphatic cells, and in the nuclei of tumor cells. These results suggest that tumor lymphatics carry specific markers and that it may be possible to specifically target therapies into tumor lymphatics.