Methotrexate regulates ICAM-1 expression in recipients of rat cardiac allografts.

The means by which methotrexate (MTX) mediates immunosuppression at low doses remains to be elucidated. MTX has been shown to inhibit the adherence of neutrophils and fibroblasts to endothelial cells in vitro. The hypothesis that MTX treatment may affect cellular adherence by downregulating cell adhesion molecule expression formed the rationale for these studies. Previous studies of rat cardiac transplant recipients in our laboratory demonstrated that low-dose MTX treatment alone significantly inhibits the expression of the leucocyte beta 2 integrin subunit, CD18. These investigations have addressed whether low-dose MTX treatment might also affect the expression of the beta-integrin counter-receptor, ICAM-1, a cell adhesion molecule which may be induced on endothelial cells during an immune response. The degree to which low-dose cyclosporine A and low-dose MTX treatment alone, and in combination, impact cell adhesion molecule expression has been studied in Brown Norway (BN) to Lewis (Lew) rat accessory cervical heart allografts. According to both Northern blot and immunohistochemical analysis, ICAM-1 expression was upregulated in graft regional lymph nodes and in the spleen of untreated cardiac allograft recipients within 6 h post-transplantation. Despite induction of VCAM-1 expression, ICAM-1 expression remained low or undetectable in cardiac allograft tissue as measured both by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical analysis. These data suggest that ICAM-1 may function in leucocyte trafficking through lymphoid organs, such as the lymph nodes and spleen, but not directly in graft leucocyte recruitment during BN to Lew rat cardiac allograft rejection. Despite prolonged allograft survival with cyclosporine A alone and combination cyclosporine A/MTX, these treatments did not result in diminished steady-state ICAM-1 mRNA levels in regional lymph nodes or spleen of cardiac allograft recipients. MTX treatment alone, however, substantially diminished ICAM-1 expression in allograft recipient lymphoid tissues. These studies demonstrate for the first time in vivo using a rat model of acute allograft rejection that MTX but not cyclosporine treatment downregulates cell adhesion molecule expression. Low-dose MTX treatment alone, however, is not sufficient to result in prolonged BN to Lew rat cardiac allograft survival. The means by which combination low-dose cyclosporine A and MTX treatment results in prolonged rat cardiac allograft survival over low-dose cyclosporine treatment alone remain(s) to be clarified.

[Retrograde infusion of prostaglandin E1 in angiologic ambulatory care: why and how?]. / Retrograde Infusion von Prostaglandin E1 in der angiologischen Ambulanz: warum und wie?

In comparison with control-groups it is shown (a) that the convenient retrograde injection makes prostaglandin E1 (PGE1) the most effective therapy for perfusion defects of different etiology and (b) that the easy application-technique and the good tolerance omits admission of these severe patients to the hospital and justifies the relatively high costs.

The resting interstitial tissue pressure in primary varicose veins.

In 31 patients with unilateral primary varicose veins the resting interstitial pressure in the deep posterior compartment of the leg was measured in three groups: I, asymptomatic; II, symptomatic; III, symptomatic with trophic changes. Spinal anesthesia was induced in all patients, and polytetrafluoroethylene (Teflon) catheters were inserted into the deep posterior compartment of both legs. The interstitial pressure was recorded with a bioelectronic strain-guage pressure monitor with the patient in the horizontal position with complete muscle relaxation. In control legs (n = 31) mean pressure was -2.48 mm Hg (range -5 to 1). However, the measurement in affected legs (n = 31) was significantly higher at 4.61 mm Hg (range 1 to 12). Results demonstrate the following: (1) Primary varicose veins (an exclusively epifascial abnormality) were consistently associated with an increased resting interstitial pressure of the subfascial tissues in the supramalleolar area of the leg. (b) The subfascial resting interstitial pressure was elevated in all stages of varicose veins, even those with short duration of the disease and with no symptoms at all. (c) In all three groups there was a significant increase in the subfascial interstitial pressure between the affected and contralateral extremity. In patients with trophic changes (group III) this increase was significantly higher than in the remaining groups, although there was no significant difference between the increased pressure of the remaining groups (groups I & II).

The role of CT in the diagnosis of primary lymphedema of the lower limb.

Twelve patients with primary lymphedema of the lower limb were examined with computed tomography (CT). A characteristic "honeycomb" pattern of the subcutaneous compartment was seen in 10 of these patients. CT scans in nine other patients with swollen leg secondary to chronic venous disease or lipedema did not show this characteristic pattern. CT may be helpful in the differential diagnosis of a swollen leg, thus obviating venography or lymphangiography.

The distribution of cholesterol and apoprotein A-I between the lipoproteins in plasma and peripheral lymph from normal human subjects.

The lipoproteins of peripheral lymph and plasma from normal human subjects were separated according to their density by sequential ultracentrifugation and according to their size by gradient gel electrophoresis and gel exclusion chromatography. High density lipoproteins (HDL) carried a higher proportion of the total cholesterol in lymph than in plasma. Within the HDL fraction, the less dense and more lipid-rich component (HDL2) carried a higher proportion of the total HDL cholesterol in lymph than in plasma. Gradient gel electrophoresis showed (1) a higher proportion of large to small HDL particles in lymph than in plasma and (2) the presence of at least three populations of apo A-I-containing lipoproteins with Stokes diameters larger than the Stokes diameter of HDL2. Separation by gel exclusion chromatography showed that the proportion of large HDL particles with a high cholester: apo A-I ratio was greater in lymph than in plasma. In view of the sieving effect of the blood capillaries, which favours the passage across the capillary walls of smaller vs larger particles, we suggest that the higher ratio of large to small HDL particles in lymph than in plasma is due to the conversion of small to large HDL in the interstitial fluid by incorporation of cholesterol and other lipids from extravascular cells into the smaller particles.

Effects of starvation and plasma exchange on lecithin: cholesterol acyltransferase activity and cholesterol efflux in cholesterol-fed pigs.

The effects of starvation and of plasma exchange with a cholesterol-free substitute on efflux of tissue cholesterol and on lecithin: cholesterol acyltransferase (LCAT) activity in plasma and peripheral lymph were investigated in two pigs fed a cholesterol diet for 3-4 months. The pigs were labelled with i.v. [14C]cholesterol before plasma exchange or starvation. The cholesterol diet increased plasma total cholesterol concentration and LCAT activity in plasma and lymph, but had little effect on the rate of esterification of cholesterol in plasma or lymph. During cholesterol feeding, and when the animals were fed a normal diet, cholesterol esterification rates in plasma and lymph were much lower than the maximum rates achieved when LCAT was saturated with substrate, suggesting that LCAT in normal pig plasma and lymph is not saturated with substrate. Plasma exchange, carried out when the specific activity of tissue cholesterol exceeded that of plasma cholesterol, was followed by a brief rise in the specific activity of plasma cholesterol to a maximum value between the specific activities of muscle and adipose-tissue cholesterol, reflecting the transfer of radioactive cholesterol from tissue to plasma. During the rise in plasma total cholesterol specific activity there were no differences between the specific activities of low-density lipoprotein (LDL) cholesterol and high-density lipoprotein (HDL) cholesterol in plasma or lymph. Starvation had no effect on the plasma-cholesterol specific-activity curve. From about day 14 after labelling, cholesterol-specific activity decreased in the order: tissues greater than lymph greater than plasma. This suggests that the transfer of cholesterol from tissues to plasma was mediated by lipoproteins in the interstitial fluid.

The effect of the canine popliteal node on the composition of lymph.

The composition of afferent lymph draining into the canine popliteal lymph node was compared with that of the efferent lymph leaving the node. Both the protein and cellular composition were studied. In twenty-five greyhounds the protein concentration of efferent lymph was greater than that of afferent lymph collected from the same limb. Although the absolute level of protein varied greatly between dogs, in a particular animal there was a constant ratio between the protein content of afferent and efferent lymph. The concentration of protein in efferent lymph was approximately double that of afferent lymph. Chromatographic analysis of lymph and the use of radio-iodinated canine albumin indicated that the reason for the increased level of protein in the efferent lymph is that the popliteal node concentrates the protein in afferent lymph. Afferent lymph contained less than 3 X 10(3) cells/ml; efferent lymph contained between 0.5 X 10(6) and 4.3 X 10(6) cells/ml, 98% of which were lymphocytes. In different dogs there was no correlation between efferent lymphocyte density and afferent or efferent protein concentration; however, when an afferent lymphatic was perfused with solutions of different protein concentration, the lymphocyte number in the efferent fluid became greater as protein concentration in the afferent perfusate was increased. The concentrating effect of the node is discussed in terms of its significance to both fluid balance and immunological surveillance.

Further evidence for the role of high density lipoprotein in the removal of tissue cholesterol in vivo.

The lipoproteins of human peripheral lymph and plasma were separated according to particle size by polyacrylamide gradient gel electrophoresis. All samples of lymph contained lipoproteins that moved to the same positions on the gel as plasma LDL and plasma HDL. Some samples of lymph also contained lipoproteins with the mobility of VLDL and IDL. The lymph lipoproteins corresponding to plasma LDL reacted with anti-LDL serum and those corresponding to plasma HDL reacted with anti-HDL serum. In the lipoprotein fraction with the mobility of HDL, the proportion of particles larger than catalase was greater in lymph than in plasma. It is suggested that the shift in size distribution towards larger HDL particles in lymph compared with plasma is due to uptake of cholesterol from extravascular tissue by HDL particles after they have reached the interstitial fluid from the plasma, rather than to preferential movement of larger particles across the capillary walls.

The concentration of apolipoprotein A-I in human peripheral lymph.

The concentration of apolipoprotein A-I in peripheral lymph of eight apparently healthy subjects has been determined by quantitative immunoelectrophoresis. Under steady-state conditions the average concentration of this apolipoprotein in lymph was 15.9 +/- 3.6 mg/dl, that is 12.24 +/- 2.3% of its concentration in plasma of the corresponding subjects. Apolipoprotein A-I could not be detected immunochemically in particles smaller than haemoglobin (Mr 67 000) when lymph was subjected to gel filtration on Sephacryl S300 superfine either by thin-layer or column modification of this method. Lymph and plasma from three subjects were fractionated by column gel filtration and apolipoprotein A-I determined by quantitative immunoelectrophoresis in delipidated fractions. It was found that the distribution of apolipoprotein A-I in lymph was shifted towards larger particles when compared to its distribution in plasma.

The effect of human peripheral lymph on cell growth in vitro.

Peripheral lymph is similar in composition to the interstitial fluid that surrounds most cells in vivo. Gel filtration is used to show that the protein composition of such lymph is considerably different from that of plasma. Primary cell cultures fail to survive in adult human plasma or serum but grow well in adult peripheral lymph collected from the dorsum of the foot. The different effects on cell cultures may be because toxic components such as low-density lipoproteins are partially filtered out by the capillary endothelium.

Evidence for the presence of tissue-free cholesterol in low density and high density lipoproteins of human peripheral lymph.

The specific radioactivity of free and esterified cholesterol in the lipoproteins of human peripheral lymph was measured in 4 normal human subjects at various intervals after labelling the tissue cholesterol by a single intravenous injection of [14C]cholesterol. The free : esterified cholesterol specific-activity ratios in lymph LDL and HDL at short and long intervals after labelling in vivo suggest that both lipoproteins were capable of acting as acceptors for tissue-free cholesterol, but that in 3 of the 4 subjects the predominant acceptor was HDL.

Concentration of lipoproteins containing apolipoprotein B in human peripheral lymph.

The concentration of apolipoprotein B (apoB) in human serum and peripheral lymph was measured by quantitative immunoelectrophoresis with anti-serum to human low-density lipoprotein. In four normal and six hyperlipidaemic subjects, total lymph apob/ml was 5-10% of total serum apoB/ml in the same subject. These ratios were equivalent to lymph apob concentrations of 60-120 microgram/ml. When the assays were carried out under conditions in which unmasking of immunoreactive sites on lymph and serum apoB was assumed to be maximal (delipidation with Nonidet P40), the lymph/serum apoB concentration ratios in three normal subjects were similar to those obtained with untreated lymph and serum.

The passage of apoproteins from plasma lipoproteins into the lipoproteins of peripheral lymph in man.

1. The transport of apoprotein B from the lipoprotein of plasma into the lipoproteins of lymph draining the foot has been studied in four men with type III hyperlipoproteinaemia. 2. Three subjects were given autologous 125I-labelled very-low-density lipoprotein (VLDL) and 131I-labelled low-density lipoprotein (LDL) by intravenous injection; the fourth was given autologous 125I-labelled VLDL and 131I-labelled intermediate-density lipoprotein (IDL) plus LDL. 3. The 125I/131I ratios in serum and lymph apoprotein B, and the 125I and 131I specific radioactivities of apoprotein B in VLDL, IDL and LDL from serum and lymph, indicate that apoprotein B in the circulating VLDL can reach peripherallymph without the intermediacy of circulating LDL.

Observations on the passage of apoproteins from plasma lipoproteins into peripheral lymph in two men.

1. The passage of radioactive apolipoproteins into lymph draining the foot was investigated in two men, each given a single intravenous injection of low-density lipoprotein containing 131I-labelled apoprotein B and of very-low-density lipoprotein containing 125I-labelled apoprotein A and apoprotein C. 2. Protein-bound 125I and 131I appeared in the lymph of both subjects. Immunoelectrophoresis of lymph lipoproteins against anti-(high-density lipoprotein) and anti-(low-density lipoprotein) showed the presence of apo-high-density lipoprotein and apo-low-density lipoprotein with faster mobilities than plasma high-density and low-density lipoprotein respectively. Most of the protein-bound 131I in lymph was recovered in the precipitin line formed by the apoprotein B-containing lipoprotein after immunoelectrophoresis. Polyacrylamide gel electrophoresis of the lymph lipoprotein fraction showed the presence of 125I-containing bands with mobilities similar to those of the apoprotein A of high-density lipoprotein and of three of the fast-moving C apoproteins. 3. These results suggest that most, if not all, of the apoproteins of plasma lipoproteins reach the interstitial fluids and that some lipoproteins undergo modification during their passage into peripheral lymph.