Cyclosporin nephrotoxicity in heart and lung transplant patients

Twenty-two patients with heart, lung or heart and lung transplants maintained on cyclosporin for periods ranging from 3 months to 10 years developed renal insufficiency which was investigated by renal biopsy. The histopathological changes were: (i) severe vascular and glomerular damage due to thrombotic microangiopathy (TM); (ii) a form of focal segmental glomerulosclerosis (FSGS); (iii) glomerular ischaemia. Rather than being separate entities, these changes appeared to represent a spectrum of pathology, some biopsies showing all three forms of glomerular injury. In all cases the glomerular changes were accompanied by arteriolar and arterial pathology, and we identified novel ultrastructural changes in the arteriolar endothelial basal lamina. Tubular atrophy was a consistent feature, the severity of which reflected the severity of the glomerular sclerosis, and which appeared to be a consequence of glomerular loss. Our findings are consistent with the nephrotoxic effects of cyclosporin being mediated chiefly via damage to preglomerular vessels and glomerular capillary endothelium. From an analysis of the clinical aspects of these cases, the effects of cyclosporin appear to be to some extent idiosyncratic, and therefore not entirely preventable, but strict monitoring of blood cyclosporin levels is essential to minimize the risk of permanent renal damage. Monitoring urinary protein in addition to plasma creatinine may detect the onset of FSGS, as proteinuria precedes creatinine elevation.      

Autoantibodies against bactericidal/permeability-increasing protein in patients with cystic fibrosis

Cystic fibrosis (CF), a genetic disorder, is characterized by chronic pulmonary infection/inflammation which leads to respiratory failure. The presence of anti-neutrophil cytoplasmic autoantibodies (ANCA) has previously been observed in the sera of patients with CF. In view of the known relationship of ANCA with primary vasculitis and of their putative pathogenetic role in these disorders, we studied the presence, specificity and isotype of ANCA and their clinical associations in 66 adult CF patients. None of the 66 CF samples had autoantibodies to the major ANCA antigens, proteinase 3 or myeloperoxidase. However, 60/66 (91%) CF samples contained IgG and 55/66 (83%) IgA, autoantibodies to bactericidal/permeability increasing protein (BPI), a recently characterized ANCA specificity. All the IgA anti-BPI-positive samples were also IgG anti-BPI-positive. The autoantibody specificity was confirmed by inhibition assay and immunoblotting of CF sera against a neutrophil granule preparation. Furthermore, in this cross-sectional study, anti-BPI levels were inversely correlated with the observed reductions in FEV1 and FVC (IgA anti-BPI and FEV1: r = 0.508, <it>p</it> &lt; 0.0001), and both IgG and IgA anti-BPI levels were higher in CF patients with secondary vasculitis (<it>n</it> = 6) than in those without (<it>p</it> &lt; 0.05). ANCA with specificity for BPI were present in the majority of CF sera in this study and autoimmune processes may be associated with the development of pulmonary injury in CF.      

TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis

To investigate the role of Toll-like receptor (TLR)9 in the immune response to mycobacteria as well as its cooperation with TLR2, a receptor known to be triggered by several major mycobacterial ligands, we analyzed the resistance of TLR9(-/-) as well as TLR2/9 double knockout mice to aerosol infection with Mycobacterium tuberculosis. Infected TLR9(-/-) but not TLR2(-/-) mice displayed defective mycobacteria-induced interleukin (IL)-12p40 and interferon (IFN)-gamma responses in vivo, but in common with TLR2(-/-) animals, the TLR9(-/-) mice exhibited only minor reductions in acute resistance to low dose pathogen challenge. When compared with either of the single TLR-deficient animals, TLR2/9(-/-) mice displayed markedly enhanced susceptibility to infection in association with combined defects in proinflammatory cytokine production in vitro, IFN-gamma recall responses ex vivo, and altered pulmonary pathology. Cooperation between TLR9 and TLR2 was also evident at the level of the in vitro response to live M. tuberculosis, where dendritic cells and macrophages from TLR2/9(-/-) mice exhibited a greater defect in IL-12 response than the equivalent cell populations from single TLR9-deficient animals. These findings reveal a previously unappreciated role for TLR9 in the host response to M. tuberculosis and illustrate TLR collaboration in host resistance to a major human pathogen.     

Guidelines for setting up an external quality assessment scheme for blood smear interpretation. Part II: survey preparation, statistical evaluation and reporting.

Blood smear analysis is a well-known technique in medical laboratories. The clinical relevance of this analysis and its interpretation are very important. Consequently, monitoring of laboratory performance by an external quality assessment scheme is strongly recommended. This article represents a very practical guidance document for External Quality Assessment Scheme (EQAS) organizers for setting up blood smear schemes. In the first part of the guidelines, the Hematology Working Group of the European External Committee for External Quality Assurance Programmes in Laboratory Medicine (EQALM) published practical information for the preparation of blood smears for use in an EQAS. Part II focuses on aspects such as survey preparation, statistical evaluation and reporting, and describes particular details for organizing blood morphology EQA surveys by means of virtual microscopy.     

兔血管内皮细胞和诱导成骨细胞在可注射纳米材料上的共培养

目的：将可注射纳米骨材料与共培养的兔肾血管内皮细胞和兔骨髓间充质干细胞诱导的成骨细胞复合,并构建可注射细胞型纳米组织工程骨,观察它们体外培养的相容性. 方法：实验于2003-09/2004-11在苏州大学附属儿童医院完成.①实验材料：取16周龄雄性新西兰大耳白兔,体质量1.5 kg左右.②实验方法：麻醉后抽取兔骨髓,用淋巴细胞分离液分离出其中的间充质干细胞,在含体积分数为0.15牛血清的RPMI1640液中培养.骨髓间充质干细胞在条件培养基中,7 d后可见细胞变为多边形,碱性磷酸酶和Ⅰ型胶原染色阳性,形成钙结节,表现成骨细胞分化特点.采用三步梯度筛网法,获得肾血管球,5 g/L胶原酶Ⅳ37℃消化15～20 min,离心沉淀获取血管内皮细胞,在含体积分数为0.15小牛血清的M199中培养.③实验评估：免疫组织化学法进行第Ⅷ因子相关抗原鉴定,透射电镜观察细胞浆Weibel-Palade小体,间接免疫荧光法检测CD31、CD34及CD44的表达.将共培养的兔肾血管内皮细胞、成骨细胞与可注射纳米骨材料体外复合培养,进行形态学观察和功能测定. 结果：①在一定培养条件下成功诱导兔骨髓间充质干细胞向成骨细胞分化.②培养的血管内皮细胞免疫组织化学法检测第Ⅷ因子相关抗原阳性,透射电镜观察到细胞浆中的Weibel-Palade小体,间接免疫荧光法检测CD31、CD34表达阳性,CD44阴性.③共培养的兔肾血管内皮细胞、成骨细胞在可注射纳米骨材料上生长、增殖良好,细胞活性和碱性磷酸酶活性未受到影响. 结论：可注射纳米骨材料具有良好的细胞相容性,可作为骨组织工程理想的可注射载体材料.     

Comparison of the roll-plate and sonication techniques in the diagnosis of microbial ureteral stent colonisation: results of the first prospective randomised study

Background

Microbial ureteral stent colonisation (MUSC) is one leading risk factor for complications associated with ureteral stent placement. As MUSC remains frequently undetected by standard urine cultures, its definitive diagnosis depends on microbiological investigation of the stent. However, a standard reference laboratory technique for studying MUSC is still lacking.

Materials and methods

A total of 271 ureteral stents removed from 199 consecutive patients were investigated. Urine samples were obtained prior to device removal. Stents were divided into four parts. Each part was separately processed by the microbiology laboratory within 6 h. Ureteral stents were randomly allocated to roll-plate or sonication, respectively, and analysed using standard microbiological techniques. Demographic and clinical data were prospectively collected using a standard case-report form.

Results

Overall, roll-plate showed a higher detection rate of MUSC compared with sonication (35 vs. 28 %, p < 0.05) and urine culture (35 vs. 8 %, p < 0.05). No inferiority of Maki’s technique was observed even when stents were stratified according to indwelling time below or above 30 days. Compared with roll-plate, sonication commonly failed to detect Enterococcus spp., coagulase-negative staphylococci (CoNS) and Enterobacteriaceae. In addition, sonication required more hands-on time, more equipment and higher training than roll-plate in the laboratory.

Conclusions

This prospective randomised study demonstrates the superiority of Maki’s roll-plate technique over sonication in the diagnosis of MUSC and that urine culture is less sensitive than both methods. The higher detection rate, simplicity and cost-effectiveness render roll-plate the methodology of choice for routine clinical investigation as well as basic laboratory research.     

Efetividade da infiltração intra-articular guiada por imagem: comparação entre fluoroscopia e ultrassom

ObjectiveCompare the effectiveness of ultrasound and fluoroscopy to guide intra-articular injections (IAI) in selected cases.Material and methodsA prospective study in our outpatient clinics at the Rheumatology Division at Universidade Federal de São Paulo (UNIFESP), Brazil, was conducted to compare the short-term (4 weeks) effectiveness of ultrasound and fluoroscopy-guided IAI in patients with rheumatic diseases. Inclusion criteria were: adults with refractory synovitis undergo- ing IAI with glucocorticoid. All patients had IAI performed with triamcinolone hexaceton- ide (20 mg/ml) with varying doses according to the joint injected.ResultsA total of 71 rheumatic patients were evaluated (52 women, 44 whites). Mean age was 51.9 ± 13 years and 47 of them (66.2%) were on regular DMARD use. Analysis of the whole sample (71 patients) and hip sub-analysis (23 patients) showed that significant im- provement was observed for both groups in terms of pain (P < 0.001). Global analysis also demonstrated better outcomes for patients in the FCG in terms of joint flexion (P < 0.001) and percentage change in joint flexion as compared to the USG. Likert scale score analyses demonstrated better results for the patients in the USG as compared to the FCG at the end of the study (P < 0.05). No statistically significant difference between groups was observed for any other study variable.Discussion and conclusionImaging-guided IAI improves regional pain in patients with vari- ous types of synovitis in the short term. For the vast majority of variables, no significant difference in terms of effectiveness was observed between fluoroscopy and ultrasound- guided IAI.     

Prevalence of intra-abdominal hypertension in critically ill patients: a multicentre epidemiological study

Objective Although intra-abdominal hypertension (IAH) can cause dysfunction of several organs and raise mortality, little information is available on the incidence and risk factors for IAH in critically ill patients. This study assessed the prevalence of IAH and its risk factors in a mixed population of intensive care patients.Design A multicentre, prospective 1-day point-prevalence epidemiological study conducted in 13 ICUs of six countries.Interventions None.Patients Ninety-seven patients admitted for more than 24 h to one of the ICUs during the 1-day study period.Methods Intra-abdominal pressure (IAP) was measured four times (every 6 h) by the bladder pressure method. Data included the demographics, medical or surgical type of admission, SOFA score, etiological factors such as abdominal surgery, haemoperitoneum, abdominal infection, massive fluid resuscitation, and ileus and predisposing conditions such as hypothermia, acidosis, polytransfusion, coagulopathy, sepsis, liver dysfunction, pneumonia and bacteraemia.Results We enrolled 97 patients, mean age 64±15 years, 57 (59%) medical and 40 (41%) surgical admission, SOFA score of 6.5±4.0. Mean IAP was 9.8±4.7 mmHg. The prevalence of IAH (defined as IAP 12 mmHg or more) was 50.5 and 8.2% had abdominal compartment syndrome (defined as IAP 20 mmHg or more). The only risk factor significantly associated with IAH was the body mass index, while massive fluid resuscitation, renal and coagulation impairment were at limit of significance.Conclusion Although we found a quite high prevalence of IAH, no risk factors were reliably associated with IAH; consequently, to get valid information about IAH, IAP needs to be measured.     

Protein tyrosine phosphatase PRL-3 in malignant cells and endothelial cells: expression and function

Protein tyrosine phosphatase PRL-3 mRNA was found highly expressed in colon cancer endothelium and metastases. We sought to associate a function with PRL-3 expression in both endothelial cells and malignant cells using in vitro models. PRL-3 mRNA levels were determined in several normal human endothelial cells exposed or unexposed to the phorbol ester phorbol 12-myristate 13-acetate (PMA) and in 27 human tumor cell lines. In endothelial cells, PRL-3 mRNA expression was increased in human umbilical vascular endothelial cells and human microvascular endothelial cells (HMVEC) exposed to PMA. An oligonucleotide microarray analysis revealed that PRL-3 was among the 10 genes with the largest increase in expression on PMA stimulation. Phenotypically, PMA-treated HMVEC showed increased invasion, tube formation, and growth factor-stimulated proliferation. A flow cytometric analysis of cell surface markers showed that PMA-treated HMVEC retained endothelial characteristics. Infection of HMVEC with an adenovirus expressing PRL-3 resulted in increased tube formation. In tumor cells, PRL-3 mRNA levels varied markedly with high expression in SKNAS neuroblastoma, MCF-7 and BT474 breast carcinoma, Hep3B hepatocellular carcinoma, and HCT116 colon carcinoma. Western blotting analysis of a subset of cell line lysates showed a positive correlation between PRL-3 mRNA and protein levels. PRL-3 was stably transfected into DLD-1 colon cancer cells. PRL-3-overexpressing DLD-1 subclones were assessed for doubling time and invasion. Although doubling time was similar among parental, empty vector, and PRL-3 subclones, invasion was increased in PRL-3-expressing subclones. In models of endogenous expression, we observed that the MCF-7 cell line, which expresses high levels of PRL-3, was more invasive than the SKBR3 cell line, which expresses low levels of PRL-3. However, the MDA-MB-231 cell line was highly invasive with low levels of PRL-3, suggesting that in some models invasion is PRL-3 independent. Transfection of a PRL-3 small interfering RNA into MCF-7 cells inhibited PRL-3 expression and cell invasion. These results indicate that PRL-3 is functional in both endothelial cells and malignant cells and further validate PRL-3 as a potentially important molecular target for anticancer therapy.