Determination of capillary leakage due to recombinant interleukin-2 by means of noninvasive conductivity measurements

Summary One of the most common side effects of treatment with recombinant interleukin-2 (IL-2) is capillary leakage. Its genesis is not completely understood. The aim of the study was to determine whether capillary leakage can be monitored by means of a non-invasive conductivity technique and to study its starting point. Eight patients with advanced renal cell cancer were studied in a medium care section of the Department of Medical Oncology, University Hospital over 4 days during treatment sessions of continuous, intravenously administered IL-2 (mean dose of 15.6 × 10⁶ IU · m^–2 · day ^–1). The fluid shift from the intravascular to the extra- and intracellular compartments was monitored by means of noninvasive conductivity measurements. Changes in blood volume were calculated from serial erythrocyte counts. The clinical parameters of capillary leakage (oliguria, positive fluid balance, and gain in mass) were recorded. The mean gain in mass was 9% after 4 days of IL-2 treatment. The extracellular fluid volume increased significantly [46 (SD 23.2)%; P < 0.01], whereas the intracellular fluid volume did not change. The increase in blood volume (BV) amounted to 7% (P < 0.05). The decline in albumin concentration was significantly more than the increase in BV [38 (SD 4.3) %; P < 0.01], indicating capillary albumin leakage. The main changes were observed after the 2nd day of treatment. From this study, it is suggested that conductivity measurements are a suitable method to monitor capillary leakage induced by IL-2, and could be used to detect the exact onset and severity of this leakage. The leakage started within the first 24 h of treatment and was detected as a fluid shift from the intravascular to the extracellular space, while the intracellular compartment remained stable. These measurements could be useful during intervention studies with the aim of preventing this adverse effect of IL-2.     

The effect of pentobarbital anaesthesia upon the extracellular fluid volume in the dog,studied by continuous infusion and single injection methods

The effect of pentobarbital anaesthesia on the volume and ionic composition of the extracellular space was studied in adult male mongrel dogs with permanent catheters in aorta and pulmonary artery. The extracellular fluid volume (Q ^ec ) was determined with: a) methods based on equilibration of the indicator throughoutQ ^ec by continuous infusion; b) methods based on the assumption that after a single injection of indicator the plasma indicator concentration equals extracellular indicator concentration as long as the log plasma indicator concentration-time curve is linear; c) a single injection method based on a closed flow system model with a single inflow and a single outflow orifice. The measurements were made before and 30 and 90 min after induction of anaesthesia. Thirty minutes after induction of anaesthesiaQ ^ec as determined with the method sub a, had decreased by about 10% and remained so during the following 60 min. The values ofQ ^ec as calculated by the method sub c fairly agreed withQ ^ec as determined with the method sub a and also showed a decrease ofQ ^ec during pentobarbital anaesthesia. The procedures sub b overestimatedQ ^ec and yielded a seemingly higherQ ^ec during anaesthesia, because the boundary conditions for these procedures do not apply. The haemoglobin concentration decreased by about 10% and the lactate concentration by about 50%. The phosphate concentration increased by about 25% while the other electrolyte concentrations (Na⁺, K⁺, Mg²⁺, Ca²⁺, Cl^–, HCO ₃ ^– ) did not change. A respiratory acidosis developed during the first 30 min and almost disappeared in the following 60 min. Possible explanations for the pentobarbital-induced concentration ofQ ^ec are discussed.     

An extracellular matrix infrastructure provides support for murine secondary palatal shelf remodelling

A cricial part of secondary palate morphogenesis is the movement of the palatal shelves from an initial vertical position on either side of the tongue to a final horizontal one above it to achieve palate closure. The immunocytochemical localization of extracellular matrix (ECM) molecules in the palatal shelf during this remodelling and reorientation revealed the existence of an ECM infrastructure within the mesenchyme. The major components of this infrastructure were collagen III, fibronectin, and hyaluronate (HA). With remodelling, HA's domain within the mesenchyme was expanded, whereas those of fibronectin and collagen III became more circumscribed. The expansion of an HA-rich matrix within the mesenchyme is thought to be crucial for palatal reorientation. The results of this study suggest that, as this expansion occurs, it is modulated by collagen and fibronectin components of the ECM infrastructure. Prior to shelf remodelling, this infrastructure may be anchored by a specialized region of the midoral epithelial-mesenchymal interface and the subjacent mesenchyme which is characterized by the unique distribution of collagen III, fibronectin, and tenascin. The midoral palatal epithelium also may play a role in directing shelf expansion. This epithelial region undergoes changes in cell packing and epithelial cell layering that correlate with shelf remodelling. These changes occur concomitantly with changes in the expression of collagen III, collagen IV, and laminin within the underlying basement membrane. The localization and patterning of tenascin within the developing palate suggests that it not only contributes to the postulated anchoring structure of the midoral epithelial-mesenchymal region, but also plays a role in the determining the fate of the medial edge epithelial cells during the final stage of palate closure.© Willey-Liss, Inc.     

The Effects of Extracellular Matrix on Tissue Engineering Construction of Cartilage in Vitro

In the study of bone and cartilage diseases in our laboratory, chondrocyte stereo-differentiation mod-el was cultured into cartilage tissue. The cultured cartilage tissue was closely similar to natural tissue inboth histological condition and biochemical metabolism. The model mimicked the chondrocyte differenti-ation and metabolism in vivo completely〔1〕. We studied some essential extracellular matrices, includingcollagen, proteoglycan(PG) and hyaluronic acid(HA) on the construction of grow…     

ERK1/2信号转导途径在低切应力上调人脐静脉内皮细胞IL-8基因表达中的作用

血管内皮细胞位于血流和血管壁之间,除受化学因素的调节外还受力学因素的影响。切应力可通过刺激相应的力学感受系统来调节内皮细胞某些基因的表达,其中包括诱导内皮细胞表达IL- 8。为了阐明MAPK信号途径中的ERK1/ 2信号通路是否参与调控低切应力上调人脐静脉内皮细胞IL- 8基因表达,采用Western blot分析了低切应力(4.2 0 dyne/ cm2 )处理不同时间内皮细胞ERK1/ 2的磷酸化水平及TPK抑制剂Genistein,MEK抑制剂PD980 5 9对其磷酸化的影响;采用定量RT- PCR检测内皮细胞经低切应力刺激或给予阻断剂后再行切应力刺激等处理后IL- 8基因的表达。结果显示:(1)低切应力处理可引起内皮细胞ERK1/ 2蛋白磷酸化水平上调,其磷酸化水平与切应力作用时间有关,具有快速、双向性的特点(在刺激10 min时达到高峰,2 h左右降至未刺激水平) ,阻断剂Genistein和PD980 5 9处理后,ERK1/ 2磷酸化水平与低切应力刺激10 min比较明显降低;(2 )阻断剂Genistein,PD980 5 9可显著抑制低切应力所致的内皮细胞IL- 8m RNA上调。结果表明低切应力可通过ERK1/ 2信号途径上调人脐静脉内皮细胞IL- 8基因的表达。     

Extracellular ionic and volume changes: The role in glia—Neuron interaction

Activity-related changes in extracellular K⁺ concentration ([K⁺]_e), pH (pH_e) and extracellular volume were studied by means ofion-selective microelectrodes in the adult rat spinal cord in vivo and in neonatal rat spinal cords isolated from pups 3–14 days of age (P3–P14). Concomitantly with the ionic changes, the extracellular space (ECS) volume fraction (α), ECS tortuosity (λ) and non-specific uptake (k′), three parameters affecting the diffusion of substances in nervous tissue, were studied in the rat spinal cord gray matter. In adult rats, repetitive electrical nerve stimulation (10–100 Hz) elicited increases in [K⁺]_e of about 2.0–3.5 mm, followed by a post-stimulation K⁺-undershoot and triphasic alkaline-acid-alkaline changes in pH_e with a dominating acid shift. The ECS volume in the adult rat occupies about 20% of the tissue, α = 0.20 ± 0.003, λ = 1.62 ± 0.02 and k′ = 4.6 ± 0.4 × 10⁻³s⁻¹ (n = 39). In contrast, in pups at P3–P6, the [K⁺]_e increased by as much as 6.5 mm at a stimulation frequency of 10 Hz, i.e. K⁺ ceiling level was elevated, and there was a dominating alkaline shift. An increase in [K⁺]_e as large as 1.3–2.5 mm accompanied by an alkaline shift was evoked by a single electrical stimulus. The K⁺ ceiling level and alkaline shifts decreased with age, while an acid shift, which was preceded by a small initial alkaline shift, appeared in the second postnatal week. In pups at P1–P2, the spinal cord was X-irradiated to block gliogenesis. The typical decrease in [K⁺]_e ceiling level and the development of the acid shift in pH_e at P10–P14 were blocked by X-irradiation. Concomitantly, continuous development of glial fibrillary acidic protein positive reaction was disrupted and densely stained astrocytes in gray matter at P10–P14 revealed astrogliosis.The alkaline, but not the acid, shift was blocked by Mg²⁺ and picrotoxin (10⁻⁶m). Acetazolamide enhanced the alkaline but blocked the acid shift. Furthermore, the acid shift was blocked, and the alkaline shift enhanced, by Ba²⁺, amiloride and SITS. Application of glutamate or gamma-aminobutyric acid evoked an alkaline shift in the pH_e baseline at P3–P14 as well as after X-irradiation. The results suggest that the activity-related acid shifts in pH_e are related to membrane transport processes in mature glia, while the alkaline shifts have a postsynaptic origin and are due to activation of ligand-gated ion channels.At P4–P6, the ECS volume was almost double that in adult rats, α = 0.37 ± 0.01 (n = 17), the ECS tortuosity was significantly higher, λ = 1.78 ± 0.02, while the non-specific uptake was not significantly different, k′ = 3.61 ± 0.56 × 10⁻³ s⁻¹. The α gradually decreased to about 24% at P12. In adult rats, electrical or adequate stimulation evoked a shrinkage of the extracellular space by 20–50%, while no significant changes in ECS volume were found in P3–P6. We conclude that the [K⁺]_e ceiling level, character of the pH_e transients, the size of the ECS volume and the activity-related ECS shrinkage are closely related to gliogenesis.     

Extracellular K+ accumulations and synchronous GABA-mediated potentials evoked by 4-aminopyridine in the adult rat hippocampus

Transient changes in extracellular potassium concentration ([K⁺]₀) and field potentials were evoked by 4-aminopyridine (4-AP; 50–100 M) and recorded with ion-selective microelectrodes in CA1b, CA3b and dentate sectors of adult rat hippocampal slices. Long-lasting field potentials recurred at a frequency of 1/60 s (0.016±0.003 Hz) in association with increases in [K⁺]₀ which were largest and most sustained in the dendritic regions where afferent fibers terminate (dentate>CAl>CA3) and in the hilus. In stratum radiatum of CA1 or stratum moleculare of the dentate these fields had a peak amplitude of 1.4±0.29 mV, duration 8.3±1.6 s, and were accompanied by increases in [K⁺]₀ of 1.8±0.22 mM that lasted 32±5.5 s (n = 17 slices). Interictal epileptiform potentials, which were brief (<0.2 s) and more frequent at 1/3 s (0.30±0.02 Hz) were also present in CA1, CA3 and the hilus and associated with small increases in [K⁺]₀ (0.5 mM, duration 2 s). Interictal activity was blocked by 6-cyano-7-nitroquinoxalone-2,3-dione (CNQX; 5–20 M); the slow, less frequent potentials were resistant to both CNQX and dl-2amino-5-phosphonovaleric acid (APV; 50 M) and reversibly blocked (or attenuated by 80%) by bicuculline methiodide (BMI) (25–100 M). The BMI-sensitive potentials were also abolished by baclofen (100 M), an effect which was reversed by 2-OH-saclofen (100 M). Focal application of KCl or GABA in the absence of 4-AP evoked long-lasting field and [K⁺]₀ potentials which were similar to those evoked by 4-AP but more sustained. The proportional relationship between the amplitudes of field and K⁺ potentials with GABA closely resembled that observed for 4-AP; in contrast the slope of KCl-evoked responses was lower. Our results demonstrate that in the adult rat hippocampus 4-AP induces in many different regions accumulations of [K⁺]₀ in synchrony with the long-lasting field potentials, which are known to correspond to an intracellular long-lasting depolarization of the pyramidal cells. These changes are smaller than those which occur in the immature rat hippocampus — which may be related to differences in Na-K-ATPase and susceptibility to seizures. These events involve the activation of GABA_A receptors, are under the modulatory control of GABA_B receptors, and likely arise from the activity of GABAergic interneurons and/or afferent terminals. The long-lasting field potentials appear to reflect mainly the direct depolarizing actions of GABA and to a much more limited extent the associated accumulation of [K⁺]₀.     

Effect of saline infusion on net reabsorption of endogenous sulfate relative to that of phosphate in intact and thyroparathyroidectomized rats

Clearance experiments have been performed to study the effects of saline infusion on the reabsorption of inorganic sulfate (SO₄) at endogenous levels. Adult female Sprague-Dawley rats on a standard diet were used. Both intact and thyroparathyroidectomized (TPTX) animals were infused with a 130 mmol/l sodium chloride solution at a low (0.15 ml/min) and a high (0.375 ml/min) rate. This increase of the infusion rate decreased the reabsorption of SO₄ in both groups of animals significantly. The fractional excretion of SO₄ in theintact rats increased from 9.9±5.6 to 18.4±3.6% (mean values±SD,p<0.001) and in theTPTX rats from 5.3±2.5 to 22.4±6.3% (p<0.001). It is concluded that endogenous parathyroid hormone has no major effect on the saline-induced inhibition of reabsorption of SO₄.This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Fr 239/9-1)