Effect of increased interstitial fluid flux on fractional catabolic rate of high molecular weight [3H]hyaluronan injected in rabbit skin

The removal rate of high molecular weight (M_r 3×10⁶) [³H]hyaluronan has been measured in rabbit paw skin in control and during increased local venous pressure induced by ligation of the femoral vein. The increased venous pressure resulted in a 20% increase of interstitial volume at 4 h after ligation, a value which relates to at least a doubling of lymph flow, i.e. also to doubling of fluid flux through the tissues. The fractional catabolic rate of hyaluronan was 0.0918±0.0170 h^-1 (n=10) and 0.0775±0.0206 h^-1 (n=11) in control and following increased venous pressure respectively (P>0.05). Since the fractional catabolic rate of hyaluronan is not affected by increased venous pressure, the removal of hyaluronan via lymphatic drainage in rabbit paw skin must be small compared to the local turnover of the polysaccharide.     

Lysogenization of Salmonella choleraesuis by phage 14 increases average length of O-antigen chains, serum resistance and intraperitoneal mouse virulence.

Three clones from a strain of Salmonella choleraesuis (serogroup C1) were lysogenized with phage 14 (P14) which converts the O-antigen of serogroup C1 salmonellae from O-6,7 to O-6,7,14. The lysogens were compared with their parental non-lysogenic clones with respect to the following properties: average length of O-antigen polysaccharide chains, sensitivity to normal human serum, and mouse-virulence. SDS-polyacrylamide gel electrophoresis of lipopolysaccharides extracted from these bacteria showed that samples from lysogens consisted mainly of long-chained molecules whereas those from non-lysogens contained mainly short-chained molecules. The O-antigen polysaccharide from a lysogen was estimated by chemical analysis to be six times as long as that from a non-lysogen. Lysogens were serum-resistant whereas non-lysogens were serum-sensitive. About 10 times more colony forming units of a lysogen than of a non-lysogen were recovered from the livers and spleens of mice on day 1 and 3 after intraperitoneal inoculation of equal doses. By comparison with S. choleraesuis, lysogenization of S. typhimurium with phage P22 or phage A4 did not affect the chain-length distribution of O-antigen polysaccharide. Our data suggest that phage 14-coded determinants increase efficiency of O-antigen biosynthesis in S. choleraesuis leading to increase in average length of O-polysaccharide chains. Increased serum resistance and mouse virulence are logical consequences of increase in average length of O-polysaccharide chains and represent phage-conferred selective advantage not previously described in Salmonella.     

透明质酸在大鼠慢性腹膜透析模型中的作用

目的:了解透明质酸在大鼠慢性腹膜透析中的作用。方法:20只雄性SD大鼠随机分为3组。高糖组(n=8)和透明质酸组(n=6)每天分别腹腔内注射25mL的4.25%葡萄糖腹膜透析液或含0.01%透明质酸(分子量为1600kD)的4.25%葡萄糖腹膜透析液,共7d;正常对照组(n=6),不进行腹腔注射。第8d进行4h腹膜功能实验并于不同时点留取血和腹透液样品进行分析。结果:透明质酸组的腹腔内液容积(IVP)显著高于高糖组(P＜0.01)。高糖组腹腔液体吸收率(K_E)显著高于正常对照组和透明质酸组;高糖组和透明质酸组的淋巴液体吸收率(K_EB)差异无显著性,均显著高于对照组。高糖组的组织间质吸收率(K_ET)显著高于对照组和透明质酸组,后两者区别不明显。结论:在大鼠慢性腹膜透析模型中多次重复使用透明质酸,可以防止由于长期使用以葡萄糖为渗透压制剂的腹膜透析液所引起的腹腔吸收率增加,增加超滤量,保护腹膜功能。     

Hyaluronan in the rat with special reference to the skin

The total hyaluronan content has been determined in rats. The animals were frozen and sectioned in a cryostat before digestion with papain and pronase. The hyaluronan content was determined by a specific radioassay and it was found that 250 g rats contained 40-60 mg of the polysaccharide. The recovery from the preparation procedure was close to 100%, as determined from tracer experiments. More than half of the hyaluronan was found in skin, approximately one quarter in the skeleton and supporting structures and less than one tenth in skeletal muscle. Based on calculated lymph flow and lymph concentration of hyaluronan, it seems that a significant fraction of the total hyaluronan in skin (greater than 1%) is removed via the lymphatics in a 24 h period. An attempt was made to isolate undegraded hyaluronan from rat skin by gentle methods giving full recovery in order to estimate the molecular weight of the polysaccharide. Hyaluronan was recovered quantitatively, but as determined from added tracer, it had been degraded. Correction for the estimated degradation gave a molecular weight of several millions for the endogenous hyaluronan.     

Borrelia burgdorferi decreases hyaluronan synthesis but increases IL-6 production by fibroblasts

Despite the prevalence of clinical data on human Lyme disease, little is known about the immunopathologic effects of the causative organism on the host. We studied the effect of Borrelia burgdorferi on hyaluronan (hyaluronic acid, HYA) production and the effect on interleukin-6 (IL-6) synthesis by cultured fibroblasts. The cell line employed in this study produced an average of 1406 ng of hyaluronan/ml within 48 h. Using both a morphological staining protocol and a quantitative radiometric assay, we noted that in the presence of a low dose of Borrelia (9.4 × 10⁵ cells/ml) the hyaluronan production decreased to an average of 1008 ng/ml, a significant difference (p < 0.05) from the amount of hyaluronan produced by the cells alone. The reduction was even more significant (p < 0.01) when a higher dose of Borrelia (9.4 × 10⁶ cells/ml) was used giving an average hyaluronan concentration of 682 ng/ml. In contrast, we found that Borrelia stimulated the cells to produce IL-6 from a baseline of 293 pg/ml to a maximal value of 842 pg/ml (p < 0.01). The spirochetes had no significant effect on cell viability, nor were we able to demonstrate invasion of the cells by the bacteria. Both a decrease in hyaluronan and an increase in IL-6 may correlate with the pathogenicity of Lyme disease in man.     

Immediate and Delayed Leukocyte Apoptosis in Two Models of Peritonitis

Leukocyte apoptosis is an energy-dependent process that facilitates resolution of the cellular inflammatory response. Levels of apoptosis can be accelerated or inhibited after exposure to various stimuli. To compare apoptosis in transmigrated leukocytes, two models of peritonitis in mice were used that both cause leukocyte influx into the peritoneal cavity: (1) intraperitoneal thioglycollate administration producing a sterile peritonitis and (2) cecal ligation and puncture (CLP) producing a polymicrobial bacterial peritonitis. Samples of blood and peritoneal exudate cells (PEC) were collected at multiple time points after induction of peritonitis. Leukocytes were either fixed immediately to determine an immediate apoptosis level or cultured for 24 h to determine a delayed apo- ptosis level. Apoptosis was assessed using terminal uridine-triphosphate nick-end labeling (TUNEL) assay, flow cytometry, and confocal microscopy. Leukocyte influx into the peritoneal cavity was confirmed in both models. At all time points, and in both models, there was increased immediate apoptosis in PEC compared with unmanipulated controls and this increase was maximal in CLP after 18 h, although it appeared to remain at a stable level in the sterile peritonitis model by 3 h. There was also an increase in PEC delayed apoptosis at early time points in both models, again maximal at 18 h for CLP, with the levels being significantly higher than the thioglycollate model at 6 h and 18 h. The mice had a relative peripheral neutropenia at 6 h after CLP, but not post thioglycollate injection, and this persisted until 42 h. Lung and liver MPO levels were elevated in CLP but did not increase after thioglycollate. There was no increase in immediate peripheral leukocyte apoptosis in either model, but an increase in delayed peripheral leukocyte apoptosis was observed by 18 h in both models. Peripheral leukocyte CD11b expression, which is a marker of activation, was also persistently elevated in the CLP model, but not in sterile peritonitis. In conclusion, CLP is a more potent stimulus for apoptosis of leukocytes than their migration to the site of inflammation alone, as occurs in the thioglycollate model. Blood leukocyte apoptosis also appears not to be dependent on CD11b expression, and therefore activation status.     

Pharmacokinetic behaviour of ACP gel, an autocrosslinked hyaluronan derivative, after intraperitoneal administration

Autocrosslinked polysaccharide (ACP) gel is a fully biocompatible cross-linked derivative of hyaluronic acid, which has prolonged in vivo residence time and improved mechanical properties with respect to native hyaluronan for use in various surgical applications. The objective of this study was to assess the pharmacokinetic behaviour of ACP gel in dogs after intraperitoneal administration. Seven beagle dogs received intraperitoneal injections of tritium-labelled ACP gel. Blood samples were taken, and urine and faeces were collected until sacrifice, scheduled at various time points from 3 to 192 h after administration. Organs were removed from the animals at autopsy. Bodily fluid and organ samples were analysed for total and non-volatile radioactivity. Non-volatile radioactivity slowly appeared in plasma, with a median T(max) of 12 h, and then declined with a mean half-life of 69 h. Total radioactivity in plasma peaked later and declined more slowly, consistent with the formation of tritiated water. Little non-volatile radioactivity was found in any organs except the liver, where about 16% of the dose was present 72 h after administration, and the intestines, where the presence of radioactivity was probably due to a retention effect. A minor amount of non-volatile radioactivity was also found in the bone marrow. In summary, ACP gel administered into the peritoneal cavity is removed slowly by active initial catabolism at the injection site, and is then catabolised by well described physiological pathway of hyaluronan degradation with final release of simple molecules such as CO(2) and H(2)O. Given its in vivo residence time, ACP gel may be considered an ideal implantable surgical device.     

Gadodiamide contrast agent 'activates' fibroblasts: a possible cause of nephrogenic systemic fibrosis

Nephrogenic systemic fibrosis (NSF) is a fibrotic disease generating intense interest due to its recent discovery, and unknown cause. It appears confined to patients with renal disease and presents as grossly thickened, indurated, tight skin that is woody to palpation. Histologically, the dermis contains thickened collagen bundles, numerous plump fibroblast-like cells, and elevated hyaluronan expression. Recent data suggest a link between the use of gadolinium chelate as an MRI contrast agent and the onset of the disease. Fibroblasts from the lesions of six NSF patients, all of whom were exposed to gadodiamide, were compared with control fibroblasts for hyaluronan and collagen synthesis. Serum from NSF patients was assessed for fibroblast hyaluronan-stimulating activity, collagen synthesis, and gadodiamide for its effect on fibroblast proliferation and matrix synthesis. NSF fibroblasts synthesized excess levels of hyaluronan and collagen compared with control fibroblasts, with up to 2.8-fold and 3.3-fold increases, respectively. NSF patient serum stimulated control fibroblast hyaluronan synthesis by up to 7-fold, and collagen synthesis by up to 2.4-fold. 1 mM gadodiamide added to culture medium stimulated fibroblast growth in a dose-dependent manner, decreasing their doubling time from 28 h to 22 h, and increasing the maximum cell density. Even a short exposure to gadodiamide stimulated cell growth, suggesting that the cells were activated by the gadodiamide. The growth of fibroblasts within contracted collagen lattices was also significantly stimulated by gadodiamide, while fibroblasts exposed to gadodiamide synthesized increased levels of hyaluronan. Control fibroblasts exposed to gadodiamide, and NSF fibroblasts exhibited an extensive pericellular coat of hyaluronan, and expressed alpha-smooth muscle actin. Gadolinium chloride did not affect fibroblast growth. This report demonstrates that NSF fibroblasts synthesize excess levels of hyaluronan and collagen, and that gadodiamide stimulates control fibroblast growth, matrix synthesis, and differentiation into myofibroblasts, suggesting a possible role for gadodiamide in the pathophysiology of NSF.     

Biochemical and pathological features of cadmium induced peritonitis in rats.

The intraperitoneal injection of cadmium chloride (1 mg/kg of the ion) twice a day at 12-hr intervals for a maximum period of 3 days produced a severe inflammatory reaction. This reaction was characterized by an increasing accumulation of the exudate, erythrocytes and inflammatory cells into the peritoneal cavity. The peak leucocyte infiltration appeared to occur in two phases. The first occurred at 48 hr and the second at 96 hr following the initial injection of cadmium. Neutrophils always constituted 48 to 56% of the total leucocytes. Infiltration of other cell types included macrophages, monocytes, lymphocytes, mast cells, eosinophils and basket cells. The cell free supernatant of the peritoneal exudate contained the highest amount of histamine at 24 hr and the lowest amount at 48 hr after the initial injection of cadmium. The supernatant also contained an increasing amount of total PGE₂ and PGF_2α and total β-glucuronidase and lactic dehydrogenase activity in relation to the time following the initial injection of cadmium. The cyclic nucleotide level in the cell free supernatant of the peritoneal exudate was not significantly altered at the early phase of inflammation. This was however, drastically increased both at 72 and 96 hr following the initial injection. In fact, a 50-fold increase in c-AMP level and 8-fold increase in c-GMP level were noted at 96 hr as compared to their respective levels at 24 hr. The data collected in the present study suggest that the release of histamine, lysosomal enzymes, prostaglandins and cyclic nucleotides are somehow involved in cadmium induced peritonitis in rats.     

Turnover of hyaluronan in synovial joints: elimination of labelled hyaluronan from the knee joint of the rabbit

After the injection of [3H]acetyl-labelled hyaluronan into normal rabbit knee joints, about 90% of its isotope content was ultimately accounted for as 3H2O. The rate of elimination of hyaluronan from synovial fluid was therefore estimated from changes in the level of 3H2O in plasma. The half-life of plasma 3H2O was 6.2 days (S.D. 0.7). As estimated from its metabolism to 3H2O, the mean intrasynovial half-life of [3H]hyaluronan of high molecular weight (modal relative molecular mass (Mr) greater than 6.0 x 10(6) was 13.2 h (range 11-15.5 h; n = 4); an otherwise identical preparation of low molecular weight (modal Mr 0.09 x 10(6] exhibited a mean half-life of 10.2 h (range 7.8-13.5 h; n = 4). The difference between the two groups was significant (P = 0.029). Both estimates were nevertheless close to those determined by others in the same species for injected proteoglycans (Mr 2.5 x 10(6), t1/2 = 12 h) and for endogenous hyaluronan calculated from changes in concentration during intravenous infusion of fluid under anaesthesia (t1/2 = 16 h). The similarity suggests that hyaluronan and proteoglycan are removed from synovial fluid by a common pathway with limited dependence on their molecular dimensions and concentrations.     

Dynamics of Blood Components and Peritoneal Fluid during Treatment of Murine E. coli Sepsis with β-1,3-D-polyglucose Derivatives

The influences of pretreatment with beta-1,3-D-polyglucose derivatives on levels of cytokines and arachidonic acid metabolites in body fluids in experimental peritonitis in mice are reported. Peritonitis was induced by an intraperitoneal injection of 10(8) live Escherichia coli. Pretreated animals survived the infection, untreated animals died about 12 h after inoculation with E. coli. Levels of IL-1 in plasma and peritoneal fluid, measured by cytotoxicity assay of the HT-2 cell line, increased significantly during the first 48 h after intraperitoneal treatment with beta-1,3-D-polyglucose-derivatized microbeads (GDM) or soluble, aminated beta-1,3-D-polyglucose (AG). After subsequent challenge with E. coli, the levels of IL-1 were significantly lower than in untreated animals. There was no increase in levels of TNF after treatment with GDM or AG, measured by cytotoxicity assay of the WEHI clone 13 cell line. After challenge with E. coli, TNF in plasma and peritoneal fluid was significantly lower compared with untreated animals. Both PGE2 and LTB4, measured by radioimmunoassay kits, were increased in peritoneal fluid after treatment with GDM and AG. After challenge with E. coli, PGE2 and LTB4 in peritoneal fluid increased to about half the concentration of infected control animals. Intraperitoneal injection of indomethacin to pretreated animals resulted in increased levels of IL-1 and TNF and decreased levels of PGE2 following challenge with E. coli. The levels of IL-1 and TNF remained elevated until the animals died after about 12 h. These studies demonstrate that the raised levels of arachidonic acid metabolites after pretreatment with GDM or AG seem to inhibit the otherwise lethal elevation of IL-1 and TNF in body fluids which is seen in untreated animals.     

Peritoneal leucocyte response to bacterial peritonitis in patients receiving peritoneal dialysis

We evaluated the quantitative peritoneal leucocyte response to antibiotic therapy in 25 CAPD patients with 57 episodes of bacterial peritonitis. Eighty-eight percent of the peritonitis episodes were initially treated with a first generation cephalosporin, but results of microbial sensitivity studies led to a change in the initial antibiotic regimen in 23 episodes. Overall, 47/57 (82%) episodes were cured by antibiotic therapy alone (responders), while 10/57 (18%) required removal of the peritoneal catheter as a curative procedure (nonresponder). Neither the duration of symptoms on initial presentation nor the status of being a nonresponder could be related to the baseline peritoneal leucocyte values, either the total (PLC) or polymorphonuclear counts (PMN). Since the baseline PLC and PMN showed a 500-fold variation, subsequent changes were expressed as a percent [PLC (%) and PMN-PLC (%)] of the baseline value. On day 3 of peritonitis, PLC (%) and PMN-PLC (%) were less in responders (26% and 10%) than nonresponders (251% and 254%) (p less than 0.001). Differentiation between responders and nonresponders based on PLC (%) and PMN-PLC (%) was associated with a high degree of sensitivity (90%) and specificity (90%). Similar results were obtained for day 4. These data suggest that the temporal pattern of PLC and PMN, when expressed as a percentage of the baseline value, may be useful in predicting those episodes of peritonitis which require removal of the peritoneal catheter.     

枸杞多糖对氯化甲基汞诱导海马神经干细胞损伤的保护作用

目的 观察分析枸杞多糖对氯化甲基汞所诱导神经干细胞(NSCs)损伤的影响.方法 取孕16 d SD大鼠胚胎的海马组织,分离纯化得到海马NSCs.将纯化后的海马NSCs增殖、生长10 d后,按随机分组方法 将其分为空白对照组(DMEM/F12培养基);枸杞多糖组(DMEM/F12培养基+枸杞多糖);氯化甲基汞组(DMEM/F12培养基+氯化甲基汞);氯化甲基汞+枸杞多糖组(DMEM/F12培养基+氯化甲基汞+枸杞多糖).分别测定各组生长环境中的海马NSCs分化、生长的情况,观察分析氯化甲基汞对海马NSCs的损伤作用,以及枸杞多糖干预后对海马NSCs的影响.结果 氯化甲基汞组和空白对照组比较,加入氯化甲基汞后的海马NSCs分化后的神经元比例(3.63%±0.62%)和神经元的周长(63.36μm ±5.57 μm)都较空白对照低;枸杞多糖组和其他各组比较,海马NSCs分化后的神经元比例(7.75%±0.59%)和神经元的周长(253.3μm ±11.21μm)都较其他各组高;氯化甲基汞+枸杞多糖组和氯化甲基汞组比较,前者生长环境下的海马NSCs分化后的神经元比例(5.92%±0.98%)和神经元的周长(111.9μm ±6.07μm)较氯化甲基汞组高.结论 氯化甲基汞对海马NSCs的分化、生长具有损伤作用;枸杞多糖可以减轻氯化甲基汞对海马NSCs的损伤作用,并能促进海马NSCs向神经元的分化及神经元的生长.     

Pharmacokinetics of piperacillin during continuous ambulatory peritoneal dialysis

We studied the kinetics of piperacillin in patients under continuous ambulatory peritoneal dialysis. Piperacillin 2 g was injected intravenously in 6 patients whereas 1 g was given intraperitoneally either a single dose in 3 patients without infection or the same dose every six-hours in 4 patients with peritonitis. Piperacillin was assayed by HPLC. After intravenous administration, the mean plasma piperacillin concentration was 3.1 +/- 5.6 mg/l at 12 h, the mean plasma t1/2 at 2.43 +/- 0.84 h, the volume of distribution at 20.4 +/- 6.3 l and the peritoneal clearance at 0.19 +/- 0.04 ml/min. After iterative intraperitoneal administration, serum and dialysate concentrations of piperacillin were above the minimum inhibitory concentration for susceptible pathogens without antibiotic accumulation. Peritoneal absorption was higher during peritonitis (83.4 +/- 4.8%) than without peritonitis (67.8 +/- 8.5%). Piperacillin 2 g IV every 8 hours or 1 g IP every 6 hours seemed to be the appropriate regimen in patients with chronic renal failure on CAPD.     

Levels of Proinflammatory Cytokines in Plasma after Pneumoccoccal Immunization in Human Immunodeficiency Virus Type 1-Infected Patients

To ascertain if immunization with pneumococcal polysaccharide vaccine is associated with rises in the levels of proinflammatory cytokines in the plasma of human immunodeficiency virus type 1 (HIV-1)-infected patients, the levels of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) were measured serially after immunization. IL-6 levels rose an average of 2.2- and 2.1-fold 6 and 8 h after immunization, respectively, but TNF-α levels remained unchanged. The levels of these cytokines were stable in unimmunized controls. Immunization with pneumococcal polysaccharide vaccine induces increases in the levels of IL-6 in the plasma of persons with HIV-1 infection.     

Tissue contents and urinary excretion of taurine after administration of L-cysteine and L-2-oxothiazolidine-4-carboxylate to rats

Tissue contents and urinary excretion of taurine were studied in rats after the administration of L-cysteine and its derivatives. Average taurine content in the liver of rats fed a 25% casein diet for 7 days increased 2-fold 2h after the intraperitoneal administration of 5 mmol of L-cysteine per kg of body weight, whereas that in rats fed a 5% casein diet for 2 days increased only slightly. The difference in the liver taurine contents between these two groups was discussed in relation to cysteine dioxygenase. Taurine contents in the heart, brain and blood did not differ significantly between these two groups or between the control and the group of rats which received L-cysteine. The increase in liver taurine concentrations after L-cysteine administration was much higher than that after L-cystine administration, suggesting a difference in their absorption. The intraperitoneal administration of 5 mmol/kg of L-2-oxothiazolidine-4-carboxylate (OTCA) resulted in a 3-fold increase in liver taurine content. The average increase in taurine excretion in the 24-h urine after OTCA administration corresponded to about 6.0% and that in the next 24-h urine to about 2.6% of OTCA administered, suggesting that nearly 10% of OTCA was metabolized to taurine and excreted in the urine.     

Catabolism of hyaluronan in rabbit skin takes place locally, in lymph nodes and liver.

The catabolism of hyaluronan has been studied by injecting hyaluronan, labelled with 125I-tyramine cellobiose (125I-TC), subcutaneously into the hindpaw of rabbits. Following endocytosis, 125I-TC remains in the cells at the site of uptake, allowing localization of the site of catabolism. At 6 h after subcutaneous injection, 65% of the injected radioactivity was recovered. The skin at the injection site contained 47%, the popliteal gland at the side of injection 10%, and the liver 8% of the injected dose. At 48 h the three organs contained 40% of the injected dose with 17% in the skin, 10% in the lymph node and 13% in the liver. The decline in recovery could be accounted for by urinary excretion of the tracer, implying that some tracer had been released from the cells after endocytosis. Chromatography revealed that over 85% of 125I-TC-hyaluronan in the lymph nodes and liver was of low molecular mass throughout the experiment. In skin, 4% of the injected tracer was recovered with low molecular mass at 6 h, increasing to 12% of injected dose at 24 and 48 h. Thus, a minimum of 12% of the injected tracer was catabolized per 24 h at the skin injection site. If cells in skin are responsible for the subsequent release of tracer, as seen from the decrease in recovery of the injected dose, another 10-15% of the tracer could have been catabolized locally in the skin per day. The major part of the hyaluronan injected in the skin was, however, catabolized by lymphatic removal and subsequent degradation in local lymph nodes and liver.     

Analysis of dimethyl sulfoxide immunosuppression in the rat model of collagen II autoimmune arthritis: An effect dependent upon intraperitoneal administration and associated with toxicity

The effect of the route of administration of dimethyl sulfoxide on humoral immunity and arthritis was evaluated in the rat model of collagen II autoimmune arthritis. Intraperitoneal administration of 5 g/kg/day (days 0–12) reduced serum anti-collagen II IgG levels, delayed the onset of arthritis, but induced sterile peritonitis in all of the treated animals. The same dose given subcutaneously did not alter humoral or clinical parameters. Lower intraperitoneal doses (0.04 and 0.25 g/kg/day), although non-toxic, were similarly ineffective. Subcutaneous (5 g/kg/day) or topical treatment (both hindpaws dipped twice daily into 70% dimethyl sulfoxide) of established disease (days 16–27) produced a mild anti-inflammatory effect without any immunosuppression. We suggest that the apparent suppression of autoimmunity by dimethyl sulfoxide is dependent upon intraperitoneal administration and a toxic dose of the agent.