Lymphatic vessels in human sural nerve: immunohistochemical detection by D2-40.

Lymphatics were detected in the epineurium of the human sural nerve by D-240 immunostaining and confirmed by ultrastructural examination.

Effects of TNF-alpha/colchicine combined treatment on Burkitt lymphoma cells: molecular and ultrastructural changes.

Tumour necrosis factor alpha (TNF-alpha) kills Daudi cells (Human Burkitt Lymphoma), inducing either necrosis or apoptosis without DNA fragmentation. Therefore, we were interested in studying the molecular and ultrastructural events occurring when the nucleus is more accessible and cells are blocked in mitosis, following colchicine treatment. In fact, as early as after 1 h treatment a typical ladder pattern was shown by means of DNA gel electrophoresis. In parallel the quantitative analysis of the different morphological patterns observed gave evidence of an increased percentage of primary necrosis after 6 h treatment, and a higher incidence of cells in late apoptosis as well as in secondary necrosis after 24 h treatment. Our findings show that Daudi cells respond to the combined treatment with an increased formation of micronuclei and nuclear alterations which follow a number of early mitochondrial changes and result in enhanced cell death. These data imply that TNF-alpha-induced apoptosis of Daudi cells can be triggered by mitochondrial changes and is somehow related to microtubule organization.

Studies on the biological effects of ozone: 9. Effects of ozone on human platelets.

During the course of ozonated autohaemotherapy (O3-AHT) using heparin as an anticoagulant, it was occasionally observed that a few clots were retained in the filter during blood reinfusion. This observation prompted an investigation on the effect of ozone (O3) on human platelets. We have now shown, both by biochemical and morphological criteria, that heparin in the presence of O3 can promote platelet aggregation. In contrast, after Ca(2+) chelation with citrate, platelet aggregation is much reduced. The potential role of the transient formation of hydrogen peroxide (H2O2) in the presence of Ca2+ with the possible expression of adhesion molecules is briefly discussed.

Catalytic competence of O-acetylserine sulfhydrylase in the crystal probed by polarized absorption microspectrophotometry.

The reactions of the pyridoxal 5'-phosphate-dependent enzyme O-acetylserine sulfhydrylase with the substrate O-acetyl-L-serine and substrate analogs have been investigated in the crystalline state by single-crystal polarized absorption microspectrophotometry. This approach has allowed us to examine the catalytic competence of the enzyme in different crystalline states, one of which was used to determine the three-dimensional structure; experimental conditions were defined for the accumulation of catalytic intermediates in the crystal suitable for crystallographic analyses.O-Acetyl-L-serine reacts with the enzyme in one of the crystal forms leading via a beta-elimination reaction to the accumulation of the alpha-aminoacrylate Schiff base, absorbing maximally at 320 and 470 nm, as in solution. The dissociation constant for the alpha-aminoacrylate Schiff base is in the millimolar range, 500-fold higher than in solution, suggesting that crystal lattice interactions may oppose functionally relevant conformational changes. The dissociation constant exhibits a bell-shaped dependence on pH centered at pH 7. At this pH the alpha-aminoacrylate species slowly decays with time (30% decrease in 24 hours). The alpha-aminoacrylate intermediate readily reacts with sodium azide, an analog of sulfide, the natural nucleophilic agent, to give a new amino acid and the native enzyme, indicating that the crystalline enzyme catalyzes the overall beta-replacement reaction as in solution. In other crystal forms, including that used for the X-ray investigation, O-acetyl-L-serine either has an even higher dissociation constant or causes crystal damage upon binding. When the crystalline enzyme reacts with either L-cysteine or L-serine, the external aldimine intermediate is formed. The dissociation constants for both substrate analogs are closer to those observed in solution and are modulated by pH as in solution. Findings demonstrate that O-acetylserine sulfhydrylase is catalytically competent in the crystal although some regions of the molecule, likely involved in an open-closed transition induced by O-acetyl-L-serine binding, may have a limited flexibility. The accumulation in the crystal of both the external aldimine and the alpha-aminoacrylate intermediate makes feasible their structural determination and, therefore, the elucidation of the catalytic pathway at the molecular level.

Immunocytochemical detection of phosphatidylinositol 3 kinase in Burkitt lymphoma cells.

PI 3-kinase, an enzyme responsible for the phosphorylation of the D3 position of the inositol ring of phosphatidylinositol (PI), is recognized to be involved in the regulation of many cellular processes such as mitogenic signalling, inhibition of apoptosis, intracellular vesicle trafficking/secretion, regulation of actin and integrin functions and regulation of protein kinases induced by tumour necrosis factor, oncoproteins and ultraviolet light. Here we report the subcellular distribution and the phosphorylative pattern of p85 alpha subunit of PI 3-kinase in Burkitt lymphoma cells exposed to R interferon alpha treatment. Immunocytochemical analysis of this enzyme, performed by confocal microscopy, revealed an increased expression of this protein at cytoplasmic level after 90 min of interferon alpha treatment. Western blotting analyses performed on nuclear and cytoplasmic fractions confirmed the overexpression found by confocal microscopy at cytoplasmic level in the 90 min interferon alpha treated cells still persisting in the 24 hr treated samples. Such an overexpression was paralleled by an increase of tyrosine phosphorylation both at cytoplasmic and nuclear level suggesting that an enhanced requirement for cytoplasmic expression and phosphorylation of PI 3-kinase might be necessary to the cell for regulating some cytoplasmic-nuclear cross talk involved in the control of Burkitt lymphoma cell metabolism following interferon alpha treatment.

Vasa vasorum of superficial collecting lymphatics of human thigh.

Collecting lymphatics were obtained from human thigh fat for light microscopy and tridimensional reconstruction at time of operation for varicose veins. No patient had lymphedema and routine sections showed no inflammation or notable pathologic alteration of the surrounding soft tissue. Abundant vasa vasorum was observed around the musculature of superficial collecting lymphatics of human thigh. Within intervalvular portions of the lymphatic collectors where the muscle coat was thicker and more compact, the vasa vasorum penetrated between smooth muscle cells and was in contact with the endothelium. In valvular portions of the collecting lymphatics where the muscle layer was thinner and more fragmented, there were fewer vasa vasorum. Tri-dimensional reconstructions of the collecting lymphatic wall showed two communicating plexi of vasa vasorum--one outside and the other inside the muscle layer. Arteries and veins of similar size did not have such an abundant vasa vasorum. The explanation for this difference may relate to the fact that a relatively low oxygen and nutrient content of lymph is insufficient to nourish the collecting lymphatic. Moreover, diffusion of nutrients from the external plexus is likely also impeded by the thickness and density of the muscle layer. The vasa vasorum deep in the muscular layer and in the subendothelial space probably sustain adequate nutrition and oxygenation to the collecting lymphatic.

An immunological correlation between the anchoring filaments of initial lymph vessels and the neighboring elastic fibers: a unified morphofunctional concept.

Little has been published on the histochemical and cytochemical properties of anchoring filaments of initial lymph vessels. Previous research suggests that the microfibrils of the anchoring filaments have ultrastructural, histochemical and cytochemical characteristics similar to those of the microfibrils associated with elastic fibers. With the aim of further investigating the histological identity of anchoring filaments, we performed an immunohistochemical study with human skin lymphatics, using antibody HB8, specific for elastic fiber microfibrils. The findings suggested strong molecular similarities between elastic fibers and the fibrils of anchoring filaments of the initial lymph vessels. A comparison of these fibrils showed both constitutional homogeneity and structural continuity from the abluminal surface of the initial lymph vessel to the perivascular elastic fibers and to the adjacent elastic network of connective tissue. In conjunction with previous findings, we propose a unified hypothesis that the elastic fiber system composed of anchoring filaments, perilymphatic sheath and adjacent connective tissue acts by alternating stretching and relaxation to propel lymph towards lymph collectors and draining lymph nodes.

Peptidergic innervation of mesenteric lymphatics in guinea pigs: an immunocytochemical and pharmacological study.

By immunocytochemistry, substance P immunoreactive (SP-IR) and vasoactive intestinal peptide immunoreactive (VIP-IR) nerve fibers were examined in guinea pig mesenteric lymph collectors. The immunoreactive nerve fibers, located in the adventitia of lymphatics, were few and were irregularly distributed along the vessel wall. These fibers appeared to be more numerous and more evenly distributed along the corresponding artery and vein walls within the same area. SP immunoreactivity in the vascular nerves was depleted in guinea pigs injected with capsaicin but was unaffected by the injection of 6-hydroxydopamine. By contrast, VIP-IR nerve fibers were unaffected by both treatments. It is concluded that SP-IR nerve fibers in the lymphatics are likely to be of sensory origin and that VIP containing nerves in the lymph collectors are distinct from SP-containing and noradrenergic nerves. It is also suggested that lymph collectors possess a complex although limited innervation pattern not only of autonomic nerve fibers containing classic neurotransmitters but also of peptidergic nerve fibers of a different origin with a vasomotor and/or sensory action.

Pathologic features in long-term cardiac allografts.

Pathologic conditions of six long-term orthotopic heart transplant survivors (11 to 17 years) was compared with a group of six similar heart transplant recipients surviving only 2 years. The two groups were matched as far as possible for age and sex of both recipients and donors and for immunosuppressive therapy (azathioprine and prednisone). Ischemic time, HLA-A, -B typing, acute rejection episodes, lipid profiles, and coronary angiograms were investigated in both groups. None of these parameters correlated with survival or disease of the graft. Graft coronary disease was present in 10 of 12 cases and caused graft failure in 8 of 12. All six long-surviving grafts and four of six surviving only 2 years showed occlusive coronary disease. The major difference in the two groups was in the pathologic condition of the coronary arteries in the long-term survivors, which more resembled that of naturally occurring atherosclerosis than the characteristic concentric graft coronary disease present in grafts surviving 2 years. Although the histopathologic features were different in the two groups, no investigated factor was useful in predicting graft disease and survival.

Morphology of the peritoneal membrane during continuous ambulatory peritoneal dialysis.

In the last 3 years we performed 52 peritoneal biopsies (PB) in 31 patients on continuous ambulatory peritoneal dialysis (CAPD). Samples of the parietal peritoneum were obtained either during insertion of the catheter or while it was being repositioned or removed. PB was performed in 13 patients before initiating CAPD and in 27 after 7-49 months of CAPD while 7 had PB during peritonitis, and, again, in 5 of these cases, PB was repeated after 1-4 months for light, electron transmission, and scanning electron microscopy. BP after CAPD showed that mesothelial cells were irregularly spaced, and at times we observed alterations in the cellular structure. Rarely were these cells degenerating, while rarefaction and in many cases complete absence of microvilli were observed. In some cases the submesothelial layers showed rarefaction of the connective tissue and sclerosis. During peritonitis, PB showed more alterations with marked degeneration and in some cases necrosis of the mesothelium and alterations of connective tissue. PB performed some months after peritonitis showed only a partial regression of these alterations and sclerotic patches, and no microvilli were noted in the mesothelium.

[Morphological study by scanning electron microscopy of the lymphatic vessels of the guinea pig]. / Studio morfologico al microscopio elettronico a scansione (SEM) dei vasi linfatici di cavia.

The purpose of this study was to describe the morphology of the whole lymphatic way: from capillaries to thoracic duct including cisterna chili using scanning electron microscopy and Evan's technique. We observed the lymph vascular wall that is: the endothelial surface, the muscular layer and the adventitial one. All these vessels were covered by an endothelial surface, with raised nuclei and long cell axes oriented parallel to the direction of flow. The borders between adjacent endothelial cell were often seen and open junctions were noted in lymphatic capillaries. The technique we used, permitted the removal of connective tissue by HC1 hydrolysis, so that smooth muscle cells could be examined. The latter showed a great variety of aspects and a very irregular course. The adventitial layer was thin in capillaries and became complex in thoracic duct where collagen fibers and connective elements were seen.

[Mesentery collector valves in the guinea pig: morpho-functional aspects]. / Le valvole dei collecttori mesenteriali di cavia: aspetti morfo-funzionali.

In guinea pig mesenterial lymph vessels there are many bicuspid valves, which determine the flow centralwards of lymph. Our observations, based on the study of 52 guinea pig lymph collectors, demonstrated a different number of valves on different parts of each vessel. In fact we found more valves in the part near the intestinal wall and the mesenterial lymph node than in the middle part of the vessel. Besides we measured the time of flow in these different portions by Indian ink injection and we found a correlation between valve number and flow. In fact the time of flow decreases with the increase of the number of valves the shortest being near the intestinal wall and the mesenterial lymph node and the longest being in the middle part of the collectors. The experimental data indicate that the valves have an important role in the lymph circulation because they favour the flow lymph velocity.

[Histochemical and ultrastructural studies of the innervation of the lymph-vessel and blood-vessel wall. 1. Adrenergic innervation]. / Richerche istochimiche ed ultrastrutturali sull'innervazione della parete vascolare linfatica e sanguifera. 1) L'innervazione adrenergica.

The adrenergic innervation of the lymph vascular wall was studied by means of the Falck fluorescence histochemical tecnique and electron microscopy with Tranzer and Richards' histochemical tecnique. The lymph vessels wall, compared with that of blood vessels, shows very few adrenergic nerve fibers located in the adventitia outside the smooth muscle cells. The possible role of the nervous system in the motor control of the lymph vessels is discussed.

[Histochemical and ultrastructural studies of the innervation of the lymph-vessel and blood-vessel wall. II. Cholinergic innervation]. / Richerche istochimiche e ultrastrutturali sull'innervazione della parete vascolare linfatica e sanguifera. II. L'innervazione colinergica.

Using the Acetylcholinesterase (AChE) tecnique applied to light and electron microscopy, was observed that the lymph vascular wall shows very few and inconstant AChE-positive fibers. The cholinergic fibers run prevalently longitudinal in the perivascular connective tissue, only brief segments show a loose network. The results are discussed and compared with blood vessels innervation.