Inhibition by uraemic sera of CD4+ T-cell adhesion to extracellular matrix components

BACKGROUND.: T-cell-mediated immune responses are impaired in patients withchronic renal failure. The migration, proliferation, differentiation,biological functioning, and interaction with other T cells aremediated by cell surface adhesion proteins, which include integrins. METHODS.: To elucidate how uraemia can impair T-cell-mediated responsesin vivo, the effects of sera from uraemic patients on T-cellproliferation and adhesion to extracellular matrix (ECM) componentswere examined. RESULTS.: Preincubation of human CD4⁺ T cells with sera from undialysedand dialysed (haemodialysis or peritoneal dialysis) uraemicpatients inhibited the capacity of the cells to be stimulatedby phytohaemmagglutinin and by anti-CD3 monoclonal antibodyplus immobilized fibronectin (FN). Sera from uraemic and dialysedpatients, but not from healthy individuals, inhibited significantly,and in a dose-dependent fashion, human CD4⁺ T cell adhesionto immobilized FN and laminin (LN). The degree of inhibitionof adhesion was similar whether the sera were continuously present,even during the adhesion assay, or removed by washing. The adhesioninhibiting capacity of the uraemic sera was not due to modificationof the expression of ß1 integrins on the surfacesof the T cells. CONCLUSIONS.: These results suggest that uraemia can impair the proliferativecapacity and adhesion of immune cells, and thus may affect normalimmune processes and contribute to the overall immune deficiencyobserved in patients with renal failure.     

Characterization of a highly polymorphic marker adjacent to the SLC4A1 gene and of kidney immunostaining in a family with distal renal tubular acidosis.

BACKGROUND: Mutations in the human SLC4A1 (AE1/band 3) gene are associated with hereditary spherocytic anaemia and with distal renal tubular acidosis (dRTA). The molecular diagnosis of AE1 mutations has been complicated by the absence of highly polymorphic genetic markers, and the pathogenic mechanisms of some dRTA-associated AE1 mutations remain unclear. Here, we characterized a polymorphic dinucleotide repeat close to the human AE1 gene and performed an immunocytochemical study of kidney tissue from a patient with inherited dRTA with a defined AE1 mutation. METHODS: One CA repeat region was identified in a phage P1-derived artificial chromosome (PAC) clone containing most of the human AE1 gene and the upstream flanking region. We determined its heterozygosity value in multiple populations by PCR analysis. Genotyping of one family with dominant dRTA identified the AE1 R589H mutation, and family member genotypes were compared with the CA repeat length. AE1 and vH(+)-ATPase polypeptides in kidney tissue from an AE1 R589H patient were examined by immunocytochemistry for the first time. RESULTS: This CA repeat, previously reported as D17S1183, is approximately 90 kb upstream of the AE1 gene and displayed considerable length polymorphism, with small racial differences, and a heterozygosity value of 0.56. The allele-specific length of this repeat confirmed co-segregation of the AE1 R589H mutation with the disease phenotype in a family with dominant dRTA. Immunostaining of the kidney cortex from one affected member with superimposed chronic pyelonephritis revealed vH(+)-ATPase-positive intercalated cells in which AE1 was undetectable, and proximal tubular epithelial cells with apparently enhanced apical vH(+)-ATPase staining. CONCLUSIONS: The highly polymorphic dinucleotide repeat adjacent to the human AE1 gene may be useful for future studies of disease association and haplotype analysis. Intercalated cells persist in the end-stage kidney of a patient with familial autosomal dominant dRTA associated with the AE1 R589H mutation. The absence of detectable AE1 polypeptide in those intercalated cells supports the genetic prediction that the AE1 R589H mutation indeed causes dominant dRTA.     

Increase in deoxyribonuclease activity in uraemic lymphocytes is caused by the cleavage of the largest polymerase I subunit

Deoxyribonucleases and DNA-dependent RNA polymerase activitiesin T and B lymphocytes isolated from patients with chronic renalfailure and control subjects were studied. The data clearlyshows that the nuclease activity in T and B cells isolated fromuraemic patients is remarkably enhanced when compared to thecontrol cells. Concomitant with the enhancement in enzyme activity,the reduction in RNA polymerase I activity and quantity wasobserved. It was found that the increase in nuclease activityand quantity was limited to the group of relatively small nucleaseswith molecular weights ranging from 14kDa to 18kDa. It has beenreported previously that these nucleases are among the cleavageproducts of the largest subunit of DNA-dependent RNA polymeraseI. Thus we suggest that the depressed metabolic activity isa characteristic feature of the uraemic lymphocyte cells andthe observed increased in DNase activity in those cells is aresult of polymerase I degradation.     

Transmembrane signalling in human monocyte/mesangial cell co-cultures: role of cytosolic Ca(2+).

BACKGROUND: Adhesion of monocytes triggers apoptosis, cytotoxicity, cytokine release, and later proliferation of cultured human mesangial cells (HMC). In the search for transmembrane signals transducing the interaction of HMC adhesion molecules with leukocyte counterreceptors, we measured variations of cytosolic Ca(2+) ([Ca(2+)](i)) in HMC and monocytes of the U937 cell line during 6-h co-cultures. METHODS: Monolayer cultures of HMC and suspensions of U937 cells were loaded with the fluoroprobe fura 2-AM and subsequently co-cultured for 6 h while separately monitoring by microfluorometry the Ca(2+)-dependent 500 nm fluorescent emission of each cell line at fixed intervals upon excitation at 340/380 nm. RESULTS: U937 and peripheral blood monocyte adhesion was followed in HMC by a slow, progressive rise of [Ca(2+)](i) from basal levels of 96+/-9 nM to 339+/-54 at 60 min and 439+/-44 nM at 3 h. The [Ca(2+)](i) elevation reached a steady state thereafter, while parallel monolayers incubated with control media maintained resting levels throughout the co-culture with stable fluoroprobe retention. Receptor sensitivity to vasoconstrictor agents, including compounds not released by monocytes, such as angiotensin II, was rapidly downregulated in HMC co-cultured with U937 cells. No [Ca(2+)](i) changes could be elicited by the octapeptide or by the TxA(2) analogue, U-46619, as early as 30 min after exposure to U937 cells. No [Ca(2+)](i) changes were observed in U937 cells throughout the co-culture. Conditioned media from monocytes and from co-cultured HMC+U937 cells had no effect on [Ca(2+)](i) of HMC. Ca(2+) entry leading to fura 2 saturation was still inducible by Ca(2+) ionophores, such as ionomycin and 4-Br-A23187, which also inhibited the responses to vasoconstrictors. Ca(2+)-free solutions prevented the [Ca(2+)](i) rise as well as subsequent receptor inactivation, implicating Ca(2+) influx through store-operated Ca(2+) channels (SOC), a major pathway for Ca(2+) entry in these cultured cells. Ca(2+) influx was confirmed by Mn(2+)-quenching of fura 2. CONCLUSIONS: In HMC, early changes in [Ca(2+)](i) signal for monocyte adhesion in a co-culture model of glomerular inflammation. This signalling mechanism may mediate the functional responses elicited in glomerular cells by leukocytes, including downregulation of receptors for vasoactive agents.     

Renal extraction of cystatin C vs 125I-iothalamate in hypertensive patients.

BACKGROUND: Several markers are available to estimate the glomerular filtration rate (GFR) in patients. Cystatin C is a relatively new marker and has been suggested as an alternative for creatinine. Numerous studies have been performed to evaluate the usefulness of cystatin C to estimate GFR. The aim of this study is to compare the renal extraction of cystatin C with that of 125I-iothalamate in hypertensive patients. METHODS: Forty hypertensive patients with unilateral renal artery stenosis, and who used at least two antihypertensive agents, were studied. For the determination of the renal extraction ratio, blood samples were drawn simultaneously from the renal vein and the abdominal aorta. The renal extraction ratio was calculated as ([A]-[V])/[A], in which A is the plasma concentration of the compound from the abdominal aorta, and V is the plasma concentration of the compound from the renal vein. RESULTS: The mean difference between the renal extraction ratio of cystatin C and that of 125I-iothalamate was 0.002. The 95% confidence interval (CI) for the mean difference was -0.036 to 0.032, which was not statistically significant. However, the limits of agreement were large (-0.271 and 0.267). CONCLUSIONS: Despite a lower reported glomerular sieving coefficient of cystatin C, the mean renal extraction of cystatin C was equal to the mean renal extraction of 125I-iothalamate in hypertensive patients, suggesting tubular secretion of cystatin C. Combined with the large variation in the renal extraction of cystatin C, these findings cast doubts on its usefulness as a glomerular filtration marker.     

Leukotriene B4 Production in Normal Rat Glomeruli

The production of the lipoxygenase metabolite of arachidonicacid, leukotriene B₄ (LTB₄) by normal rat glomeruli has beenstudied. Isolated glomeruli from saline-perfused rat kidneyswere incubated in Kreb's buffer for 15 mm at 37°C. The concentrationsof LTB₄ and other eicosanoids in cell-free supernatants weredetermined by direct radioimmunoassays (RIA). Mean basal synthesesof eicosanoids were: thromboxane B₂ (TXB₂ 0.77±0.13,prostaglandin (PG) E₂ 0.40±0.05, 6-keto-PGF₁ 0.38±0.06ng/mg glomerular protein (mean±SEM n=8). In these assaysimmunoreactive LTB synthesis was 0.12±0.02 ng/mg. Insamples incubated with BW75SC, 50 µg/ml, an inhibitorof arachidonate metabolism via both the cyclo-oxygenase andlipoxygenase pathways, there was more than 80% inhibition ofthe synthesis of TXB₂, PGE₂ and 6-keto-PGF₁. However, immunoreactiveLTB₄ was only reduced by 44%, suggesting the presence of othermaterials which cross-react in the RIA. The presence of authenticLTB₄ in the supernatants was confirmed after extraction andhigh-pressure liquid chromatography (HPLC). This material represented25% of the original material detected by RIA. Although the physiologicalrole of LTB₄ in the normal state is unknown, its chemotacticactivity may be of great significance during glomerular inflammation.     

2,3 Diphosphoglycerate-haemoglobin binding in uraemic patients treated with erythropoietin. A 31P-nuclear magnetic resonance study

Using ³¹P-nuclear magnetic resonance (NMR) measurements of relaxationrate for 2,3 diphosphoglycerate (DPG) phosphorus atoms, we showedpreviously that in uraemic red blood cells the DPG-haemoglobinbinding is stronger, thus stabilizing the deoxyhaemoglobin formand hence facilitating oxygen release. Here we verified if thesemodifications of spatial environment of DPG remain in uraemicpatients treated by human recombinant erythropoietin (rHuEpo).Simultaneously we measured the intraerythrocytic ATP concentration(ATP₁) and pH (pH₁) of patients. Our results show a slight decreaseon pH₁ and ATP₁ values during rHuEpo treatment. For the DPGrelaxation rates, we observed a very weak but statisticallysignificant increase 6 months after the beginning of treatment,but we cannot attribute a physiopathological significance tothese results because of the lack of accuracy of the NMR determinationof relaxation rate in red blood cells. Therefore, the DPG-haemoglobin binding is always stronger thanin normal subjects.     

Comparability of Narcotrend index and bispectral index during propofol anaesthesia

Background. The dimensionless Narcotrend^TM (NCT) index (MonitorTechnik,Germany, version 4.0), from 100 (awake) to 0, is a new indexbased on electroencephalogram pattern recognition. Transferringguidelines for titrating the Bispectral Index^TM (BIS, AspectMedical Systems, USA, version XP) to the NCT index depends ontheir comparability. We compared the relationship between BISand NCT values during propofol anaesthesia. Methods. Eighteen adult patients about to have radical prostatectomywere investigated. An epidural catheter was placed in the lumbarspace and electrodes for BIS and NCT were applied as recommendedby the manufacturers. After i.v. fentanyl 0.1 mg, anaesthesiawas induced with a propofol infusion. After intubation, patientsreceived bupivacaine 0.5% 15 ml via the epidural catheter. Forty-fiveminutes after induction, the propofol concentration was increasedto substantial burst suppression pattern and then decreased.This was done twice in each patient, and BIS and Narcotrendvalues were recorded at intervals of 5 s. The efficacy of NCTand BIS in predicting consciousness vs unconsciousness was evaluatedusing the prediction probability (P_K). Results. We collected 38 629 artefact-free data pairs of BISand NCT values from the respective 5-s epochs. Because of artefacts,another 5008 epochs had been excluded from data analysis (3855epochs for the NCT index alone, 245 epochs for the BIS aloneand 908 epochs for both indices). Mean (SD) values in awakepatients were 94 (6) for Narcotrend and 91 (8) for BIS. Withloss of the eyelash reflex, both values were significantly reduced,to 72 (9) for NCT (P<0.001) and to 77 (11) for the BIS index(P<0.001). The P_K value for loss of eyelash reflex was similarfor BIS (0.95) and NCT (0.93). Decreasing BIS values coincidedwith decreasing NCT values. A sigmoid model [NCT index=52.8+26.8/(1+exp(–(BIS–78.3)/4.8))^0.4;r=0.52] described the correlation between BIS and NCT indexin a BIS range between 100 and 50. For BIS values lower than50, a second sigmoid model with a correlation of r=0.83 wasapplied [NCT index=6.6+45.3/(1+exp(–(BIS–29.8)/2.4))^0.6 r=0.83]. The relationship between burst suppression ratio(BSR) and NCT index was best described by the following sigmoidmodel: NCT index=265/(1+exp((–BSR+108)/–49); r=0.73. Conclusions. We found a sufficient correlation between BIS andNCT index, but deviations from the line of identity in someranges require attention. Therefore, a simple 1:1 transfer fromBIS to NCT values is not adequate. Our results might serve asa blueprint for the rational translation of BIS into NCT values.     

Morphological and metabolic changes in the cortex of mice lacking the functional presynaptic active zone protein bassoon: a combined 1H-NMR spectroscopy and histochemical study

Mice lacking functional presynaptic active zone protein Bassoon are characterized by an enlarged cerebral cortex and an altered cortical activation pattern. This morphological and functional phenotype is associated with defined metabolic distortions as detected by a metabonomic approach using high-field (14.1 T) high-resolution 1H-nuclear magnetic resonance spectroscopy (MRS) in conjunction with statistical pattern recognition. Within the cortex but not in the cerebellum, concentrations of N-acetyl aspartate, glutamine, and glutamate are significantly reduced, whereas the majority of all other detectable low molecular metabolites are unchanged. The reduction of the neuron-specific metabolite N-acetyl aspartate in the cortex coincides with a significant decrease in neuronal density in cortical layer V. Comparing the neuron with glia cell densities across the cortex reveals cortex layer-dependent alterations in the ratio between both cell types. Whereas the ratio shifts significantly toward neurons in the cortical input layers IV, the ratio is reversed in cortical layer V. Consequently, the previously observed altered neuronal activation pattern in the cortex is reflected not only in defined cytoarchitectural anomalies but also in metabolic disturbances in the glutamine-glutamate and N-acetyl aspartate metabolism.     

Effect of chronic uraemia on skeletal muscle metabolism in man

Fatigue and lethargy, common symptoms in uraemia, have beenattributed to many factors. To assess possible bioenergeticcontributions to this, we examined the forearm muscle of fivepatients in end-stage renal failure using ³¹P-magnetic resonancespectroscopy. There was a small increase in the ratio of intracellularinorganic phosphate to ATP in resting muscle, suggesting anincreased cytosolic phosphate concentration. During exercise,increased phosphocreatine breakdown was accompanied by rapidintracellular acidification and an increase in calculated lacticacid accumulation in the muscle of the uraemic subjects, suggestingglycolysis dominating over oxidative phosphorylation as a sourceof ATP. After exercise, the half-time of phosphocreatine (PCr)recovery was longer in the uraemic subjects, suggesting diminishedmitochondrial function. The initial rate of PCr resynthesiswas not significantly decreased, but when account was takenof the high cytosolic ADP concentration (which drives mitochondrialoxidative ATP synthesis) the calculated maximum oxidative capacitywas significantly reduced in the uraemic subjects. Thus therewas evidence of mitochondrial dysfunction in uraemia due eitherto limitation of oxygen supply, reduced mitochondrial content,or an intrinsic mitochondrial defect. This resulted in increasedphosphocreatine depletion and increased glycolytic ATP productionduring exercise and there was partial compensation of the mitochondrialabnormality by increased ADP concentration. In three of thesepatients studied after elevation of haemoglobin with erythropoeitin(from 8 to 12g/dl), initial phosphocreatine breakdown and lacticacid accumulation during exercise were normalized, while exerciseduration and calculated maximum oxidative capacity remainedsignificantly abnormal. This suggests that anaemia contributesto these metabolic abnormalities but does not fully explainthem.     

Brain networks associated with cognitive reserve in healthy young and old adults

In order to understand the brain networks that mediate cognitive reserve, we explored the relationship between subjects' network expression during the performance of a memory test and an index of cognitive reserve. Using H2(15)O positron emission tomography, we imaged 17 healthy older subjects and 20 young adults while they performed a serial recognition memory task for nonsense shapes under two conditions: low demand, with a unique shape presented in each study trial; and titrated demand, with a study list size adjusted so that each subject recognized shapes at 75% accuracy. A factor score that summarized years of education, and scores on the NART and the WAIS-R Vocabulary subtest was used as an index of cognitive reserve. The scaled subprofile model was used to identify a set of functionally connected regions (or topography) that changed in expression across the two task conditions and was differentially expressed by the young and elderly subjects. The regions most active in this topography consisted of right hippocampus, posterior insula, thalamus, and right and left operculum; we found concomitant deactivation in right lingual gyrus, inferior parietal lobe and association cortex, left posterior cingulate, and right and left calcarine cortex. Young subjects with higher cognitive reserve showed increased expression of the topography across the two task conditions. Because this topography, which is responsive to increased task demands, was differentially expressed as a function of reserve level, it may represent a neural manifestation of innate or acquired reserve. In contrast, older subjects with higher cognitive reserve showed decreased expression of the topography across tasks. This suggests some functional reorganization of the network used by the young subjects. Thus, for the old subjects this topography may represent an altered, compensatory network that is used to maintain function in the face of age-related physiological changes.     

Strong depletion of CD14(+)CD16(+) monocytes during haemodialysis treatment.

BACKGROUND: The immune defect in haemodialysis (HD) patients is associated with a monocytic dysfunction, including an increased production of proinflammatory cytokines. Monocytes fall into subpopulations comprising CD14(++)CD16(-) and CD14(+)CD16(+) cells. Circulating numbers of the latter can rapidly increase during infectious episodes and inflammation. METHODS: We determined the amount of CD14(+)CD16(+) monocytes in HD patients and characterized their fate during HD treatment. In 34 HD patients and 17 healthy controls, the distinct cell populations were determined by differential blood counts and flow cytometry. Cells from 14 HD patients were analysed at the start, 10, 30 and 120 min thereafter, and at the end of HD treatment. RESULTS: Before HD, patients show a monocytosis with a strongly increased CD14(+)CD16(+) subpopulation. Early during HD treatment, circulating leukocyte numbers decrease, with monocytes being most profoundly influenced. Interestingly, among them, sequestration is most pronounced in the CD14(+) CD16(+) subpopulation. After 30 min, approximately 83+/-9% of CD14(+)CD16(+) cells are removed from circulation. This sequestration does not differ between patients treated with polyamide or haemophan membranes. The sequestration is a short-lived temporary effect and cell numbers are replenished within 120 min of treatment for the entire monocyte population. Beyond that time point, cellular activation by the dialyser membrane becomes visible. Reappearence kinetics of CD14(+)CD16(+) monocytes is slower; however, initial numbers are reached by the end of treatment. CONCLUSION: Haemodiaysis leads to temporary removal of monocytes from the bloodstream followed by the reappearance of activated cells. This might contribute to the state of chronic microinflammation, which is reflected by high levels of CD14(+)CD16(+) monocytes.     

Blunted renal dopaminergic system activity in puromycin aminonucleoside-induced nephrotic syndrome.

BACKGROUND: A primary tubular sodium handling abnormality has been implicated in the edema formation of nephrotic syndrome. Dopamine synthesized by renal proximal tubules behaves as an endogenous natriuretic hormone by activating D(1)-like receptors as a paracrine/autocrine substance. METHODS: We examined the time courses of the urinary excretion of sodium, protein and dopamine in puromycin aminonucleoside (PAN)-treated and control rats. The rats were sacrificed during greatest sodium retention (day 7) as well as during negative sodium balance (day 14) for the evaluation of renal aromatic l-amino acid decarboxylase (AADC) activity, the enzyme responsible for the synthesis of renal dopamine. Also, the influence of volume expansion (VE) and the effects of the D(1)-like agonist fenoldopam (10 microg/kg bw/min) on natriuresis and on proximal tubular Na(+),K(+)-ATPase activity were examined on day 7. RESULTS: The daily urinary excretion of dopamine was decreased in PAN-treated rats, from day 5 and beyond. This was accompanied by a marked decrease in the renal AADC activity, on days 7 and 14. During VE, the fenoldopam-induced decrease in proximal tubular Na(+),K(+)-ATPase activity was more pronounced in PAN-treated rats than in controls. However, the urinary sodium excretion during fenoldopam infusion was markedly increased in control rats but was not altered in PAN-treated animals. CONCLUSION: PAN nephrosis is associated with a blunted renal dopaminergic system activity which may contribute to enhance the proximal tubular Na(+),K(+)-ATPase activity. However, the lack of renal dopamine appears not to be related with the overall renal sodium retention in a state of proteinuria.     

22 CD4+CD25+调节性T细胞在肝癌微环境中的分布状况与局部免疫状态的关系

笔者对52例肝癌组织和癌旁组织用CD4和CD25双重酶标免疫组化染色标记Treg和CD4+T细胞，对20例肝癌组织和癌旁组织用CD8EnVision法染色标记CD8+T细胞，光镜下计数阳性细胞，对癌组织中Treg细胞和CD4+T,CD8+T及CD4+T/CD8+T值进行相关性分析，并用正常肝组织作对照。结果示8例正常肝组织中未发现Treg细胞。52例肝癌、癌旁组织中Treg细胞单个高倍视野平均数分别为7.6308±2.8368和5.1654±1.6718（P＝0.000）;肝癌中Treg细胞数目与肿瘤大小、有无子灶、有无癌栓及肿瘤临床病理分期有关，组间差异有显著性（P<0.05），而与患者年龄、性别、有无肝硬化、术前AFP浓度、肿瘤有无包膜及肿瘤Edmondson分级无明显关系（均P>0.05）;肝癌中Treg细胞数量与其浸润性CD4+T淋巴细胞的数量以及CD4+T/CD8+T值呈显著负相关（r=-0.539，P＝0.014;r=-0.545，P＝0.000），而与浸润性CD8+T淋巴细胞的数量分布无关（r=-0.403 P＝0.078）。提示肝癌微环境中的Treg细胞可能通过抑制肿瘤局部免疫，使肿瘤细胞逃避免疫监视，促进肿瘤的进展以及侵袭或转移;肝癌组织中的Treg细胞数量可能作为判断肝细胞癌预后的指标之一。