Lipid level and type alter stearoyl CoA desaturase mRNA abundance differently in mice with distinct susceptibilities to diet-influenced diseases.

Chronic diseases develop in susceptible individuals following exposure to environmental conditions including high fat diets. Inbred strains of mice differing in susceptibility to atherosclerosis, diabetes, obesity and certain cancers are models for understanding the genetic basis and molecular mechanisms whereby diet influences these polygenic and multifactorial disorders. Expression sequence tags (EST) and disease quantitative trait loci (QTL) are also being identified with these strains. Reported here are comparisons of food intake, growth, nonfasting serum lipids and expression of mRNA for hepatic apolipoprotein E (ApoE), hepatic stearoyl CoA desaturase (Scd1) and heart lipoprotein lipase (Lpl) in a 2 x 2 x 2 design with C57BL/6J and BALB/cByJ mice fed semipurified diets with 4 or 20% saturated (coconut) or unsaturated (corn) oils for 4 mo. Histological studies of aortas and coronary arteries are also reported for these animals. After 4 mo, BALB/cByJ mice were significantly heavier and had significantly higher total serum cholesterol, HDL cholesterol and triglyceride concentrations in the fed state than C57BL/6J mice. Efficiency of utilizing dietary energy did not differ consistently between strains. Oil level affected serum total cholesterol, triglycerides and HDL cholesterol, which were significantly greater in mice fed high fat diets. Lpl and ApoE mRNA expression levels were not significantly affected by mouse strain, oil source or oil level. Scd1 mRNA expression, however, was significantly higher in C57BL/6J than in BALB/cByJ mice and was lower in all mice fed 20% compared with those fed 4% fat diets. Genes regulated differently by diet among strains with distinct susceptibility to diet-influenced disease may be associated with molecular pathways contributing to incidence or severity.

Lymphatic system of the rat pancreas.

Light and electron microscopy combined with morphometric analysis were used to investigate the distribution, extent and structure of lymphatic vessels in the head, body and tail of the rat pancreas. Serial sections 3-4 microns in thickness were cut from tissue fixed by perfusion. Alternate sections were processed for light microscopy. Intervening sections were left uncovered to be re-embedded and sectioned for electron microscopy as needed. Vessels with valves were tentatively identified as lymphatics using the light microscope, with final identification being made on adjacent sections by electron microscopy. The ultrastructure of the pancreatic lymphatic vessels was typical of lymphatics generally. Interlobular lymphatic vessels were present throughout the pancreas and were found to be associated primarily with blood vessels lying in connective tissue septa. Intralobular lymphatics were also seen but were comparatively rare. Only about 19% of the wall of the lymphatic system of the pancreas was in close relationship to acinar cells--none was closely related to the endocrine islets. The mean volume density of the system was 0.0012 microns3/microns3 and the profile density of lymphatics was 3.24/mm2. Special attention was paid to the areas of contact between adjacent endothelial cells. Open gaps of more than 30 nm in width were rare. Dilatations and associated cytoplasmic processes, suggestive of a type of intercellular transport, were seen in addition to the intracellular cytoplasmic vesicular system. The findings are consistent with the view that the lymphatic system of the pancreas does not have a specific role in the transport of pancreatic secretions other than the removal of macromolecules that may escape to the interstitium in small amounts under normal circumstances. The fine structure of the endothelial wall suggests that the mechanism of lymph formation in the pancreas is more comparable to that in other encapsulated organs such as the kidney and liver than to that in the dermis or diaphragm where fluid appears to enter lymphatics primarily by way of gaps between adjacent cells.

Lymphatic valves of the rat pancreas.

The detailed structure of pancreatic lymphatic valves in rats was examined in an attempt to identify ultrastructural features that could be correlated with the ability of these delicate structures to withstand retrograde flow. Sprague-Dawley rats were perfusion-fixed and the pancreas processed for light and electron microscopy. Lymphatic vessels were identified by their typical appearance coupled with the presence of valves within their lumen. These valves were consistently formed of cuspid leaflets joined to the lymphatic wall at the bases and sides enclosing valvular pockets or sinuses between cusp and wall. Each cusp or leaflet consisted of two simple squamous endothelial layers separated by a connective tissue core and thus appeared, at first sight, as a simple infolding of the lymphatic vessel lining with its underlying connective tissue. However, certain differences were seen. Frequently the free margins of the cusps, instead of being smooth as might be expected, exhibited endothelial extensions or processes which were arranged in such a way that they could interdigitate with similar extensions on the opposing cusps and thus aid in closure of the valves. A striking difference between the endothelial lining of the vessel and that of the cusp was the presence of a distinct and almost continuous basal lamina underlying the endothelial cells which lined the surface of the cusp facing the valve pocket. The opposing surface of the cusp, which faced the central lumen was similar to the typical lining of lymphatics in showing little or no basal lamina.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in lymphatic and blood capillary permeability: ultrastructural-functional correlations.

The major structural features of lymphatic capillaries, as they contrast with blood capillaries and as they pertain to endothelial permeability, are reviewed briefly with special emphasis on intrarenal vessels. The most characteristic structural feature of lymphatic endothelium is the discontinuity of the basal lamina. Basal laminae of blood vessels, such as renal glomerular capillaries, are prominent and are known to play a role in preventing extravasation of plasma proteins. By analogy, the lack of a basal lamina around lymphatic capillaries can be considered to be of major functional importance in facilitating access of interstitial macromolecules to the abluminal surface of endothelial cells and thus to the transport pathways that provide entry to the lymph. Tracer studies with horseradish peroxidase, for example, reveal that the protein enters the intraendothelial cytoplasmic vesicular system suggesting that this system may provide a transport pathway. Tracer is also seen between adjacent endothelial cells but in the kidney, liver and thyroid these intercellular channels comprise relatively narrow spaces of about 20 nanometers or less and do not form prominent gaps such as are seen in lymphatics of the diaphragm and skin. Evidence that macromolecular transport across endothelial cells may be asymmetric, favoring movement from interstitium to lymph, is derived from 1) studies using isolated perfused lymphatics, 2) differential luminal and abluminal membrane staining with cationic stains, 3) the presence of charged microdomains on lymphatic endothelial cell surfaces revealed with macromolecules of different charges, and 4) studies on cultured monolayers of porcine arterial endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphatic system of the thyroid gland in the rat.

Intraglandular thyroid lymph vessels in the rat were studied by qualitative and quantitative analyses in order to obtain information regarding their structure, distribution, relationships, and possible mechanisms of lymph formation. Ultrastructurally, the lymphatic vessels were similar to those described in other organs. The volume density of the lymphatic vessels was 0.007, the profile density 5.68 mm2 and the maximum diameter 17.87 nm. Ultrastructurally, visible transport pathways across the vessels appear to be represented by intracytoplasmic vesicles and channels between endothelial cells. The mean maximum diameter of the vesicles was 96 nm and they occupied 6.9% of the cytoplasm. They were equally distributed between luminal, abluminal, and intracytoplasmic positions. Open junctions (greater than 30 nm) were not seen between endothelial cells, but dilations along part of the length of interdigitating and overlapping contacts were frequent. It was concluded that the mechanism of lymph formation in the thyroid is similar to that in the kidney and liver, but differs from that in the dermis or diaphragm. However, the volume density of the vesicles of the thyroid was twice that of the liver and more than twice that of the kidney. This finding is consistent with an increase in transendothelial vesicular transport of macromolecules.

The renal cortical lymphatic system in the rat, hamster, and rabbit.

Rat, hamster, and rabbit renal cortical lymphatics were examined by light and electron microscopy. Rat and hamster kidneys possessed both intra- and interlobular lymphatics that were structurally similar at the light microscopic level. Ultrastructural examination of the hamster lymphatic endothelium, however, revealed an unusual arrangement of cytoplasmic extensions not seen in the other two species. The intralobular lymphatics were related primarily to tubules, afferent arterioles, and renal corpuscles and were consistent with lymph formation from both plasma filtrate and tubular reabsorbate. Interlobular lymphatics were seen in connective tissue associated with the interlobular blood vessels. Rabbit cortex contained only interlobular lymphatics. Cross-sectional area, maximum diameter, volume density, and profile density were determined by stereological measurements using a computer-based image analyzer. The morphological data from the rat were used, in combination with published values for lymph flow, to calculate the rate of lymph formation per unit area of endothelium in lymphatics of the renal cortex. Among kidneys fixed by retrograde perfusion, the cortical lymphatic system was most extensive in maximum diameter, volume density, and profile density. It was smallest in the rabbit and intermediate in the rat. Lower volume and profile density were found for rat kidneys fixed by the dripping technique. It was concluded that: tubular reabsorbate probably contributes to renal lymph in the rat and hamster, but not in the rabbit; significant differences exist in the extent of the renal lymphatic systems among the three species, with the hamster kidney having the richest network and the rabbit the poorest; the method of fixation influences the measured size and density of renal cortical lymphatics; and the estimated rate of lymph formation in the kidney of the rat is roughly comparable to that in the dog.

Distribution of charged sites on lymphatic endothelium.

The charge distribution on the luminal and abluminal aspects of fixed and living lymphatic endothelium was examined with particular emphasis on the endocytotic vesicular system and interendothelial junctions. Native ferritin (NF; pl = 4.5), when administered abluminally to perfused lymphatics, entered endocytotic vesicles and abluminal and luminal caveolae; NF was also found in intercellular channels, in contrast, NF when applied luminally was largely excluded from both luminal caveolae and intercellular channels. Cationic ferritin (CF; pl = 8.4) bound to the discontinuous basal lamina and to the abluminal plasma membrane, clustering preferentially around the stomata of abluminal caveolae. CF did not, however, bind to the plasma membrane of, or enter, either the vesicular system or intercellular channels, when administered abluminally. When added to the perfusion fluid CF bound to the luminal membrane and to the infundibula of intercellular channels. Ruthenium red (RR) and alcian blue (AB), both cationic stains, bound intensely to the luminal membrane and much less so to the abluminal surface, thus simulating the binding pattern of CF. Unlike CF, however, RR and AB bound to the membranes of abluminal and luminal caveolae with the same level of staining as to the plasma membrane to which they were attached. These results reflect a marked asymmetry in the membrane charge characteristics of endothelial cells.

Temperature dependence of protein transport across lymphatic endothelium in vitro.

The purpose of the work was to develop an in vitro model for the study of lymphatic endothelium and to determine, using this model, whether or not a cytoplasmic process may be involved in transendothelial transport. Segments of canine renal hilar lymphatics were dissected clean, cannulated at both ends, and transferred to a perfusion chamber for measurement of transendothelial protein transport and for ultrastructural tracer studies. The segments were subsequently processed for light and electron microscopy. By both structural and functional criteria the lymphatics were judged to have retained their integrity. At 37 degrees C, 36 lymphatics showed a mean rate of protein transport of 3.51 +/- 0.45 (SEM) micrograms/min per cm2 of lymphatic endothelium. The rate was influenced by the temperature of the system, being significantly reduced by 49% +/- 4.8, 31% +/- 5.3, and 29% +/- 3.9 when the temperature was lowered to 4 degrees, 24 degrees, and 30 degrees C, respectively. When the temperature was raised to 40 degrees C, the rate was significantly increased by 48% +/- 12.2. The vesicular system and the intercellular regions in vessels with increased or reduced rates of transport were analyzed quantitatively to ascertain whether the rate changes could be correlated with ultrastructurally demonstrable changes in either of these postulated pathways. No significant changes in junctional or vesicular parameters were found between the control lymphatics and those perfused at 24 degrees, 30 degrees, and 40 degrees C. At 4 degrees C, the temperature at which the rate of protein transport was maximally reduced, vesicular size decreased, and the number of free cytoplasmic vesicles increased, whereas the number associated with the abluminal and luminal surfaces decreased. We concluded that isolated perfused lymphatic segments transport protein at a relatively constant rate under control conditions, and that this transendothelial transport comprises both temperature-dependent and temperature-independent mechanisms. The findings were considered in terms of the different theories of lymph formation and were interpreted as providing support for the vesicular theory.

Esophageal cytology in patients receiving cytotoxic drug therapy.

Our experience with the cytology of esophageal brush specimens from patients who did not have either gastric or esophageal cancer but who received a variety of chemotherapeutic agents is reported. Ten patients received chemotherapy alone, and nine received combined chemotherapy and radiation therapy. Of the ten patients, three showed evidence of moderate to severe epithelial atypia, which was not seen in the combined treatment group or the control group. The cytologic atypias in the three patients consisted of variation in nuclear size with crowding and overlapping, an increased nuclear-cytoplasmic ratio and multiple nucleoli, many of which varied in size and shape. There was also evidence of cell death and keratinization. Although these patients had not been irradiated, other striking changes were similar to those seen following radiation therapy. The cytologic changes reported here pose a serious diagnostic problem because they may be so severe as to mimic malignant transformation. Evidence of infection by herpes simplex alone or herpes simplex associated with Candida was seen in 32% of the treated patients and in 4% of the control group.

Fine needle aspiration biopsy of thyroid nodules.

The clinical value of the fine needle aspiration of thyroid nodules was evaluated by comparing preoperative cytology to subsequent pathology in 109 patients undergoing thyroidectomy. Preoperative cytology was reported as insufficient cellular material (31 patients), benign goiter (27 patients), follicular neoplasm (22 patients), thyroiditis (12 patients), suspicious for papillary carcinoma (nine patients), Hurthle cell neoplasm (five patients), medullary carcinoma (one patient), lymphoma (one patient), and metastatic adenocarcinoma (one patient). Operative findings demonstrated that the overall sensitivity of fine needle aspiration in diagnosing thyroid neoplasia (carcinoma or adenoma) was 88% and its specificity was 80%. Operation verified the cytologic diagnosis of medullary carcinoma, lymphoma, metastatic adenocarcinoma, and seven of nine papillary carcinomas. Of the five patients with an aspiration biopsy diagnosis of Hurthle cell neoplasm, three patients had carcinoma and one had an adenoma. Four carcinomas and 12 follicular adenomas were found in patients with a cytologic diagnosis of follicular neoplasm. Thyroiditis was confirmed at operation in all 12 patients with this diagnosis on fine needle aspiration. One carcinoma was found in the 27 patients with benign goiter diagnosed on cytology. Fine needle aspiration is a valuable tool that can lead to earlier diagnosis and treatment of thyroid cancer. However, a negative aspiration does not supplant good clinical judgement in determining the need for thyroidectomy.

The organization of endocytotic vesicles in lymphatic endothelium.

The lymphatic endothelium from renal hilar lymphatics in the rat was subjected to qualitative and quantitative ultrastructural analysis with emphasis on the extent and disposition of its vesicular component. The uncoated endocytotic vesicles had an average maximum diameter of 0.073 micron and occupied 7% of the cytoplasm. There were approximately 21 vesicles in each cubic micrometer of cytoplasm. In standard electron microscopic preparations of glutaraldehyde-fixed endothelium 50% of the vesicles appeared to lie free within the cytoplasm. The remainder were seen to touch or open onto the luminal or abluminal surface of the endothelium. The degree to which intracytoplasmic endocytotic vesicles remained discrete or communicated with the plasma membrane was examined using tannic acid and ruthenium red. These substances specifically bind to charged molecules on the cell surface and identify membranes continuous with it. When this technique was applied to aldehyde-fixed tissue nearly 90% of the vesicles that were apparently free within the cell could be shown to retain a connection with the surface, approximately equal numbers communicating with either the luminal or abluminal surface. At least 15% of these vesicles existed as intercommunicating clusters. These results suggest that many vesicles are not simple discrete units, but rather form parts of chains that reach either luminal or abluminal surface. Thus, apparently discrete vesicles may be parts of vesicular chains cut in cross section. The possible relation between chemical fixation and this plan of vesicular organization is discussed and it is concluded that while chemical fixation may result in an overestimation of the numbers of intercommunicating vesicles, the qualitative aspects of vesicle disposition seem largely unaffected. Although the functional significance of our observations has yet to be determined, should this plan of vesicular organization apply to initial lymphatics as well, the concept of vesicular transport solely by random movement of discrete vesicles across lymphatic endothelium should be modified.

Effect of experimental glomerulonephritis on the cells in canine renal lymph with special reference to the veiled cell.

An immunological glomerulonephritis was induced in dogs by the administration of rabbit anti-canine glomerular serum (1 mg/kg) and the effects on white cells in blood, thoracic duct lymph and peripheral renal lymph were observed over 14 days. The principal response was seen in the renal lymph which showed, within 1 hr of the injection of antiserum, a significant increase in its cellular content that lasted for approximately 16 hr. The cells affected by this early response were phagocytic, being both mononuclear and polymorphonuclear. Although the number of lymphocytes leaving the kidney did not increase, the character of these cells changed. Thus, during the first day there was a comparative increase in the number of large lymphocytes, and cells undergoing mitosis appeared in the lymph. These changes suggested the presence of local blast transformation. Later in the course of the response the proportion of small lymphocytes increased and that of large lymphocytes declined. Non-lymphocytic mononuclear cells with characteristics of 'veiled' and 'frilly' cells appeared in renal lymph on the first day and persisted throughout the 14 days. Many of these cells formed the centres of lymphocyte rosettes. The presence of these cells, which have previously been associated with Langerhans cells in the skin, in renal lymph suggests that they have a wide distribution in the body and that they are important during the immunological response to glomerulonephritis.

Concentration of renin in the renal interstitium, as reflected in lymph.

The relationship between renin activity in renal venous plasma and in renal interstitial fluid, as reflected in the hilar lymph, was observed. Control measurements in 28 dogs demonstrated that renin levels in the hilar lymph were 3.5 +/-- (SEM) 0.4 times higher than in renal venous plasma and 6.1 +/- 1.0 times higher than in arterial plasma. Renin activity was increased to varying levels by raising ureteric pressure and by the administration of different doses of furosemide. Under all conditions, the changes in renin activity in renal venous plasma and in the interstitium, as reflected in the hilar lymph, were in parallel although often marked in the hilar lymph. Thus, the study shows that under conditions of increased renin release, the interstitial renin activity rises in consort with increases in arteriovenous plasma differences. This increase in interstitial renin activity occurred even under circumstances when renal blood flow is known to increase, thereby suggesting that angiotensin generated in the interstitium may have little effect on the cortical arteriolar caliber.

A simultaneous comparison of the cells of blood, renal hilar and thoracic duct lymph in the dog.

A simultaneous morphological and quantitative profile was obtained of the cells of blood, thoracic duct, and renal hilar lymph in the dog. Monolayer cytocentrifuged preparations were used to determine the number, type, and size of cells in the three compartments. The cell count of renal lymph was not related to that of blood or thoracic duct lymph. There was a greater percentage of lymphoid cells in the afferent lymph than could be accounted for by the random movement of cells from the blood to the lymph. Thus, there appeared to be a selective transit of cells from blood to lymph. Monocytes and neutrophils were largely absent from the thoracic duct lymph; however, eosinophils were present. Cells were observed in hilar lymph that were characteristic of cells subjected to antigenic stimulation. It was concluded that lymphocytes have a preferential pathway from blood to lymphatic and in the course of this pathway they undergo a change which is consistent with an active immunological role.