Postnatal development of lymphatic vessels and their smooth muscle cells in the rat diaphragm: a confocal microscopic study

This paper reports on how lymphatic vessels and their smooth muscle cells develop in the diaphragm of postnatal rats. Lymphatic endothelial cells in the diaphragm were labeled by an intraperitoneal injection of DiI-labeled acetylated low-density lipoprotein (DiIac-LDL). During postnatal week 1, DiI-ac-LDL was detected in many free cells in addition to distinct endothelial cells that formed lymphatic vessels. Occasionally, saccular lymphatics isolated from previously formed lymphatics were recognized; these were referred to as lymphatic islands. The DiI-ac-LDL-labeled free and lymphatic endothelial cells showed immunoreactivity for CD 34 and Flt-4, but most of them did not express either OX 62 or ED 1 immunoreactivity, with only some showing ED 1 immunoreactivity. This suggests that most of the DiI-ac-LDL-labeled elements were lymphatic endothelial cells, and that some were macrophages. After postnatal week 1, the DiI-ac-LDL positive cells were restricted to lymphatic vessels. Until postnatal week 6, lymphatic vessels increased as the diaphragm enlarged. Towards the end of postnatal week 2, free cells expressing alpha-smooth muscle actin (alpha-SMA) immunoreactivity increased in the diaphragm, and some of these were in contact with lymphatics. A coarse plexus of smooth muscle cells surrounding the lymphatic vessels first appeared at postnatal week 2, and this plexus became denser with age. Our findings indicate that lymphatic vessels are formed not only by sprouting from previously formed lymphatic vessels but also by migrating endothelial cells, and that smooth muscle cells may be differentiated from mesenchymal cells to form a plexus surrounding the lymphatic vessels.     

The three-dimensional microvascular arrangement around rat dorsal hairs as revealed by scanning electron microscopy

The three-dimensional microvascular arrangement around the dorsal hairs in vascular corrosion casts of adult Wistar rats was studied by scanning electron microscopy. Each anagen dorsal hair was surrounded by a basket-like capillary network, which was supplied by the branches of the subcutaneous artery and drained into the veins continuous with the subcutaneous vein. The capillary network surrounding the anagen dorsal hair was denser at its lower part, and became more sparse at its upper part. Transmission electron microscopy showed that the capillaries around the hair bulb possessed fenestrations. Our findings indicate that the microvascular arrangement around the anagen dorsal hair is so organized as to supply the hair bulb, which is the most important area for hair growth, with abundant blood. This study was presented at the 24th Annual Meeting of the Clinical Electron Microscopy Society of Japan, Okayama, September 17–19, 1992.     

Quantitation of varicella-zoster virus DNA in patients with Ramsay Hunt syndrome and zoster sine herpete

Varicella-zoster virus (VZV) reactivation causes facial nerve palsy in Ramsay Hunt syndrome (RHS) and zoster sine herpete (ZSH) with and without zoster rash, respectively. In the present study, we analyzed the VZV DNA copy number in saliva samples from 25 patients with RHS and 31 patients with ZSH using a TaqMan PCR assay to determine differences in the viral load between the two diseases. VZV copy number in saliva peaked near the day of the appearance of zoster in patients with RHS. Consequently, VZV DNA was less frequently detected in patients with RHS who exhibited facial palsy several days after the appearance of zoster. These findings suggest that the VZV load in saliva samples reflects the kinetics of viral reactivation in patients with RHS. In addition, VZV DNA was equally detected in saliva from patients with RHS and ZSH, and there was no significant difference in the highest viral copy number between patients with RHS and those with ZSH. The VZV load does not appear to reflect a major difference between RHS and ZSH.     

Origins and pathways of fluid entering sublobular lymphatic vessels in cat livers

The liver, which produces a large volume of lymph, has a lymphatic system which can be classified into three categories: portal, sublobular, and superficial lymphatic vessels. As little is known about the origin and pathways of sublobular lymph, this study demonstrates pathways of interstitial fluid flowing into sublobular lymphatic vessels. Livers from cats whose thoracic ducts were either ligated or non-ligated were examined by light-, transmission electron- and scanning electron-microscopy (SEM). Complete ligation of the thoracic duct caused significant dilation of the hepatic sinusoids, the space of Disse, and channels passing through the limiting plate. Sublobular interstitial space and sublobular lymphatic vessels were also expanded. The channels between hepatocytes forming the limiting plate contained collagen fibers, and connected the space of Disse with a sublobular interstitial space. The alkali-water maceration/SEM confirmed that collagen fibers traversing the layer of the limiting plate independently of blood vessels connected collagen fibers in the space of Disse with those in the sublobular space. Complete ligation of the thoracic duct also showed an accumulation of mast cells and plasma cells in the sublobular interstitial space. Our data suggest that fluid in the space of Disse flows along collagen fibers in channels traversing the limiting plate as well as those along the sinusoids and central veins that drain into sublobular veins, and enters the sublobular interstitial space to finally lead into sublobular lymphatic vessels. Our study has also shown that hepatic lymphostasis causes the accumulation of mast cells and plasma cells in the sublobular interstitial space, which may be involved in lymphangiogenesis and fibrogenesis.     

Novel TSC2 mutation in a patient with pulmonary tuberous sclerosis: lack of loss of heterozygosity in a lung cyst

A Japanese patient with tuberous sclerosis (TSC), who manifested with multiple lung cysts and pneumothorax, is described. All exons of two TSC genes, TSC1 and TSC2, in peripheral blood leukocytes from the patient were analyzed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). A novel T-to-G transition was found in exon 19 of TSC2 at nucleotide position 2168. This mutation caused an amino acid change, L717R. There was no such mutation in any other family members or in 100 normal Japanese. An automated sequencer-assisted quantitative analysis of normal and mutated SSCP-bands revealed no loss of heterozygosity (LOH) in the lung cyst tissue of the patient.     

Multinucleated cells formed on calcified dentine from mouse bone marrow cells treated with 1α,25-dihydroxyvitamin D3 have ruffled borders and resorb dentine

Osteoclast-like multinucleated cells were formed from mouse bone marrow mononuclear cells, and their morphology on coverslips and on calcified dentine slices was compared by means of transmission electron microscopy. Addition of 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] to bone marrow cells cultured on coverslips greatly stimulated the formation of multinucleated cells within 8 days. These multinucleated cells had the cytological features of osteoclasts (abundant pleomorphic mitochondria, a large number of vacuoles and lysosomes, many stacks of Golgi membranes, and an extensive canalicular system), but they developed neither ruffled borders nor clear zones. The multinucleated cells appeared to result from direct fusion of mononuclear progenitor cells, whose structural features were similar to those of multinucleated cells. Like isolated osteoclasts, both multinucleated cells and their precursors exhibited an intense reaction for tartrate-resistant acid phosphatase (TRACP) in the cisterns of endoplasmic reticulum and lysosomes. Multinucleated cells formed from alveolar macrophages in the presence of 1α,25(OH)₂D₃ were totally negative for TRACP reaction. When marrow cells were cultured on dentine slices in the presence of 1α,25(OH)₂D₃, some of the multinucleated cells were located in the shallow resorption lacunae of dentine surfaces, and they developed the characteristic ruffled borders and clear zones. The narrow extracellular spaces of the ruffled borders, the adjacent pale endocytotic vacuoles, and the dark lysosomes located in the perinuclear cytoplasm of the multinucleated cells contained numerous apatite crystals delete in resorption lacunae. These results indicate that (1) the multinucleated cells formed on coverslips from mouse marrow cells treated with 1α,25(OH)₂D₃ exhibit non-functional basic features of osteoclast morphology, and (2) differentiation of the multinucleated cells into functional osteoclasts requires some components of calcified dentine.     

Functional mapping of the activity of the R region in the human T-cell leukemia virus type I long terminal repeat to increase gene expression

We previously demonstrated the activity of the R fragment in the long terminal repeat of human T-cell leukemia virus type I for elevation of the level of gene expression. In this study, the fragment was deleted with BAL31 nuclease to determine its functional domain. Series of the shortened R fragments were linked to the simian virus 40 promoter unit, which regulated expression of a reporter gene. Examination with the R fragments deleted from the 5 and 3 ends showed that borders of the functional domain were mapped within nucleotide positions 458 to 473 for the 5 end and nucleotide positions 559 to 594 for the 3 end, respectively. Thus we conclude that a 136-base-pair fragment corresponding to the second half of the R region was sufficient to allow elevation of the level of gene expression.     

Invasive fungal infection following reduced-intensity cord blood transplantation for adult patients with hematologic diseases.

Invasive fungal infection (IFI) is a significant complication after allogeneic hematopoietic stem cell transplantation (HSCT); however, we have little information on its clinical features after reduced intensity cord blood transplantation (RICBT) for adults. We reviewed medical records of 128 patients who underwent RICBT at Toranomon Hospital between March 2002 and November 2005. Most of the patients received purine-analogbased preparative regimens. Graft-versus-host disease (GVHD) prophylaxis was a continuous infusion of either tacrolimus 0.03 mg/kg or cyclosporine 3 mg/kg. IFI was diagnosed according to the established EORTC/NIH-MSG criteria. IFI was diagnosed in 14 patients. Thirteen of the 14 had probable invasive pulmonary aspergillosis and the other had fungemia resulting from Trichosporon spp. Median onset of IFI was day 20 (range: 1-82), and no patients developed IFI after day 100. Three-year cumulative incidence of IA was 10.2%. Four of the 13 patients with invasive aspergillosis (IA) developed grade II-IV acute GVHD, and their IA was diagnosed before the onset of acute GVHD. The mortality rate of IFI was 86%. Multivariate analysis revealed that the use of prednisolone >0.2 mg/kg (relative risk 7.97, 95% confidence interval 2.24-28.4, P = .0014) was a significant risk factor for IA. This study suggests that IFI is an important cause of deaths after RICBT, and effective strategies are warranted to prevent IFI.     

Role of N-methyl-D-aspartate receptors and the nitric oxide pathway in nociception/hyperalgesia elicited by protease-activated receptor-2 activation in mice and rats

Activation of the peripheral protease-activated receptor-2 (PAR-2) triggers nociceptive behaviour and thermal hyperalgesia in rats. The present study created a novel mouse model for PAR-2-triggered nociception, and then examined the roles of NMDA receptors and the nitric oxide (NO) pathway in nociceptive processing by PAR-2. Intraplantar administration of the PAR-2 agonist SLIGRL-NH(2) elicited nociceptive responses in mice, an effect being more specific in mast cell-depleted mice. This PAR-2-triggered nociception was abolished by the NMDA receptor antagonist MK-801, but not the neuronal NO synthase inhibitor 7-nitro indazole. In contrast, the PAR-2-triggered thermal hyperalgesia in rats was blocked by both agents. Our study thus provides a novel mouse model for PAR-2-mediated nociception, and suggests that NMDA receptors are involved in PAR-2-triggered nociception and hyperalgesia, while NO contributes only to the latter.