Journal of Laboratory and Clinical Medicine最新文献

Primary IgA nephropathy, a chronic nephritis with variable prognosis, is characterized by mesangial immunoglobulin A, frequently with codeposition of other immunoglobulin isotypes and complement components accompanying matrix expansion typically preceding glomerular scarring. Glomerular immunoglobulin G, when present, is localized to the mesangial periphery found variably in repeat biopsies. IgG anti-mesangial cell autoantibodies (IgG-MESCA) in sera of patients with IgA nephropathy, specific by F(ab′)₂ binding to 48- and 55-kD autoantigen(s) could account for these deposits, but their in vivo localization, and the functional role in promoting scarring is unknown. A specific monoclonal antibody raised previously to these human mesangial cell autoantigen fractions, in this study localized to similar glomerular sites, reinforcing the view that immunoglobulin G deposition in vivo is a result of antibody–autoantigen binding. The propensity for immunoglobulin G more than other isotypes to enhance inflammation prompted study of its functional role in vitro. Using cultured human mesangial cells in a complement-free tritiated glycosaminoglycan synthesis single outcome assay, purified IgG fractions from patient sera increased matrix production in a dose-dependent manner compared with controls. At a constant total IgG concentration, matrix synthesis was proportional to the titre of IgG-MESCA. Autoreactive IgG stimulated matrix synthesis when compared with controls or IgA fractions. These findings are consistent with IgG-MESCA autoantibodies enhancing mesangial matrix synthesis in vitro, which suggests that in IgA nephropathy, similar prosclerotic autoimmune mechanisms might operate. Recombinant TGFβ₁ also induced matrix synthesis, raising the possibility that both autoimmune mechanisms and those TGFβ₁-dependent are functional or inter-related. The pathogenesis of glomerular scarring and loss in IgA nephropathy may include, in part, these mechanisms.

Background: Elevated white blood cell (WBC) count on admission in patients with ST segment elevation myocardial infarction (STEMI) has been associated with an adverse prognosis. Whether successful reperfusion by primary percutaneous coronary intervention (PCI) is associated with a decrease in WBC count is unknown. Methods: In this subanalysis of the On-TIME trial, WBC count was measured on admission and 6 h and 24 h after primary PCI for STEMI (n = 364). Angiographic measurements of reperfusion, including TIMI-flow and myocardial blush grade, were compared with changes in WBC count. Results: Restoration of TIMI 3 flow by primary PCI was associated with a significant decrease in median WBC count (11.5 (9.7–14.2), 10.7 (9.0–12.5), 9.9 (8.5–11.5) at baseline, 6 h and 24 h), whereas after unsuccessful PCI (TIMI < 3 flow) WBC count remained elevated (12.5 (9.5–14.6), 12.1 (9.9–14.4), and 11.4 (9.2–15.2)). Improved myocardial blush was also related to a decrease in WBC count. After multivariate analysis, improved myocardial perfusion (TIMI 3 flow and myocardial blush grade 3) was an independent predictor of a decrease of WBC count after PCI. Conclusion: Impaired myocardial reperfusion after primary PCI for STEMI is associated with persistent WBC elevation.

Background: Whole blood provides one of the most common sources of both high-quality DNA and high-quantity DNA for molecular biological purposes. Typically, blood storage at 4°C is short term, which ranges from a few days to a few weeks. However, long-term storage usually involves blood being frozen, with a resultant loss in DNA yield. The authors examined the effects of long-term storage at 4°C. Methods: Duplicate blood samples were collected from 301 participants (aged 20–98 years) enrolled as part of ongoing studies. Samples were stored at 4°C for between 11 days and 922 days, and DNA was subsequently extracted using a phenol/chloroform procedure. Results: A negative correlation of the number of storage days existed at 4°C with DNA yield. The main determinant on DNA yield was the age of the participant in the study, with older persons having a lower DNA yield. Conclusions: Long-term storage of blood at 4°C does have a detrimental effect on DNA yield, but this effect seemed less significant than the age of a person. The impact of age of a person or storage time has a minimal impact on DNA quality. Therefore, storage of blood at 4°C offers an acceptable alternative to frozen storage.

Intra- and extracellular lipid accumulation and the production of inflammatory mediators by renal and accessory cells may play an important role in the initiation and progression of these lesions. Platelet activating factor (PAF) is a biologically active phospholipid that is produced by various cells upon activation by different stimuli. It has been suggested that PAF plays a role in atherogenesis, and several studies indicated its participation in the pathogenesis of renal diseases. The aim of this study is to investigate the role of PAF on intracellular lipid accumulation and gene regulation of lipoprotein receptors in human mesangial cells (HMCs). A human mesangial cell line (HMC) was used to study the effects of PAF on foam cell formation by Oil red O staining and on the expression of LDLr, SR-AI, and PAF-R mRNA using RT-PCR. Native LDL caused foam cell formation in HMC in the presence of PAF. PAF enhanced LDLr expression and overrode LDL receptor suppression induced by a high concentration of LDL. Moreover, it enhanced SR-AI expression. PAF also caused increase in PAF-R expression. The above data suggest that PAF enhances its own receptor expression and then increases lipid accumulation by dysregulating LDL receptor regulation and inducing scavenger receptor expression in HMCs. These results suggest that PAF has a potential role in lipid mediated renal injury.

Integrin is the major adhesion molecule for the attachment of podocytes to the glomerular basement membrane, and integrins have been shown to play a major role in the regulation of cell survival. In this study, the authors investigated the apoptosis and its related signal pathways to integrin in cultured rat podocytes. Apoptosis was detected with the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) technique. Cytochrome c was examined by immunohistochemical stain, and Fas, Fas ligand, Bax, Bcl-2, and ERK activation (p-ERK/ERK) were analyzed by Western blotting analysis. The results demonstrated that the integrin antagonist, Gly-Arg-Gly-Asp (GRGD), increased the percentage of cells with apoptosis (from 0.9±0.5% to 27.2±9.9%, P < 0.01). Inhibition of protein tyrosine kinase with genistein also caused apoptosis of podocytes (from 0.9±0.5% to 26.0±8.7%, P < 0.01). In GRGD-treated cells, cytochrome c was found released into cytoplasm by immunohistochemical study and the Bax expression was upregulated, whereas Bcl-2 expression was not changed. Fas was not expressed in both control and GRGD-treated podocytes, although Fas ligand was upregulated in GRGD-treated cells. ERK activation was also found to be increased in GRGD-treated cells. The results indicated that α3β1integrin is necessary for the prevention of the apoptosis of cultured rat podocytes, and that the signaling involves the Bax, Bcl-2, and cytochrome c pathways.

Background: Inflammation and endothelial dysfunction are prominent in preeclampsia. Microparticles (MPs) may link these processes, as MPs induce the production of pro-inflammatory cytokines by endothelial cells and cause endothelial dysfunction. Aim: To study changes in expression of inflammation-related genes in human endothelial cells in response to MPs from preeclamptic patients. Methods: Human umbilical vein endothelial cells (HUVECs) were incubated for various time intervals in the absence or presence of isolated MP fractions from preeclamptic patients (n = 3), normotensive pregnant women (n = 3), non-pregnant controls (n = 3), and interleukin (IL)-1α as a positive control. Total RNA was isolated and used for multiplex ligation-dependent probe amplification (MLPA) and real-time polymerase chain reaction (PCR). Results: IL-1α enhanced the expression of IL-1α, IL-2, IL-6, and IL-8; nuclear factor of kappa light chain enhancer in B-cells (NFκB)-1, NFκB-2, and NFκB-inhibitor; cyclin-dependent kinase inhibitor and monocyte chemotactic protein-1; and transiently increased tissue factor expression. RNA expression of inflammation-related genes and genes encoding adhesion receptors, however, were unaffected by any of the MP fractions tested. Conclusion: MLPA is a suitable assay to test the inflammatory status of endothelial cells, because incubation with IL-1α triggered substantial changes in RNA expression in endothelial cells. Taken together, it seems unlikely that MPs from preeclamptic patients induce endothelial dysfunction by directly affecting the expression of inflammation-related genes in these cells.

Chromosomal rearrangements involving the telomeres are implied as a significant cause of idiopathic mental retardation. The most frequently used technique to detect these rearrangements was fluorescent in situ hybridization (FISH), an expensive and labor-intensive technique. One of the most promising alternative techniques is multiplex ligation-dependent probe amplification (MLPA). Here, the authors present the evaluation of a double set of probes (the SALSA P036, P019, and P020 human telomere test kits) on a series of 95 patients and 22 normal controls. Overall, 34 patients had been studied by telomeric FISH and MLPA, which was demonstrated to be a reliable method to detect essentially all subtelomeric rearrangements characterized by FISH. In addition, in these 34 patients, 13 dose imbalances were detected by MLPA, but not by FISH analysis. Overall, 12 alterations were observed only with one of the two sets, and they corresponded to polymorphic variants, as they were inherited from healthy parents or also appear in normal controls. The remaining 61 patients were initially studied with SALSA P036, and any putative dose alteration was confirmed with the two other kits and FISH. In the whole series, the authors found 9 dose imbalances evidenced with 2 MLPA kits and confirmed by FISH, representing 10% of patients with subtelomeric rearrangements. On the other hand, one small duplication at 14q11 may be clinically relevant as it appears de novo in one patient. In conclusion, MLPA can be considered a quick, sensitive, cost-effective, and easy method to screen for subtelomeric rearrangements, but any finding based in the testing of one probe should be confirmed by other sources.