Determinants of blood flow and ultrafiltration in continuous arteriovenous haemodiafiltration: theoretical predictions and laboratory and clinical observations

In continuous arteriovenous haemofiltration (CAVH) or haemodiafiltration (CAVHD), it is important to obtain an adequate blood flow through the haemofilter to minimise the risk of excessive haemoconcentration and clotting. In this study we determined the resistance to blood flow of the extracorporeal device as well as the hydraulic permeability of the filter membrane is intensive care patients treated with CAVHD. Data were obtained for CAVH catheters and Scribner shunts and for a polyacrylonitrile (AN-69) plate filter, an AN-69 capillary filter and a polysulphone (PS) capillary filter. In accordance with recent literature we also predicted the resistance to flow by using Poiseuille's law and a formula for the estimation of blood viscosity. Although with all three filters an adequate blood flow was usually obtained, the resistance to blood flow was 2-3 times greater than the predicted value. With continued use of the filter, resistance to blood flow remained largely unchanged. When, in the laboratory, the AN-69 capillary filter was perfused with saline and with a viscous sucrose solution, the resistance to flow was only 1.4 time the predicted value, a difference that might result from small deviations of the capillary diameter. When perfused with blood, the resistance was 2.6 times greater than the predicted value. This was largely explained by gross underestimation of blood viscosity in these patients. By combining laboratory data on filter resistance during saline perfusion and a more accurate estimation of blood viscosity, a reasonably accurate prediction of blood flow rate would be feasible. In the clinic the hydraulic permeability of the filters decreased with time.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet-Associated IgG in Thrombocytopenia: A Comparison of Two Techniques

The results obtained in the analysis of 130 thrombocytopenic patients with a radioimmunoassay (RIA) for platelet-associated IgG (PA-IgG) and the platelet suspension immunofluorescence test (PIFT) were compared. The RIA was positive in 33 of 41 (82.9%) patients with idiopathic thrombocytopenia (ITP) and in 51 of 79 (64.4%) patients with secondary thrombocytopenia (STP). The PIFT was positive in 37 of the 41 (90.2%) ITP patients and in 57 of the 79 (72.2%) STP patients. Sensitivity and specificity for the diagnosis of ITP of both tests were comparable: 82.9 and 40.9% for the PA-IgG(RIA) and 90.2 and 36.7% for the PIFT. A significant positive correlation was observed between the mean amount of PA-IgG measured and the height of PIFT scores with anti-IgG. Of 38 discrepancies between PA-IgG(RIA) and PIFT with anti-IgG, 15 were due to borderline results, 17 were associated with abnormal platelet-size distribution and 20 were associated with occurrence of IgM antibodies. These results suggest influences of platelet fragments and/or aggregates on accurate measurement of PA-IgG. Both fragments and aggregates escape from accurate platelet counting, while their contribution to the total IgG content remains. Therefore, a falsely elevated PA-IgG (RIA) may be measured.     

Differential expression of cell cycle and apoptosis related proteins in colorectal mucosa, primary colon tumours, and liver metastases

AIMS: Tumour cell growth results from a disturbance in the balance between the rate of proliferation and cell death. In this study, proteins involved in the regulation of cell cycle arrest and apoptosis were studied as possible factors responsible for uncontrolled cell growth in colorectal cancer. METHODS: The expression of proteins involved in these processes was investigated in 48 metastases from patients with colorectal cancer and compared with eight normal colon mucosa samples and 14 primary tumours. Both primary tumours and metastases were obtained from eight patients. The expression of thymidylate synthase (TS), p53, retinoblastoma protein (Rb), Fas receptor, Fas ligand, bcl-2, mcl-1, bax, and bcl-x was measured using immunohistochemistry. Proliferation was determined by Ki67 staining, whereas apoptosis was assessed by M30 immunostaining, which recognises cleaved cytokeratin 18. RESULTS: In the limited number of cases in which paired comparisons were possible, the expression of TS and Ki67 was significantly higher in metastases than in the matched primary tumour samples (p = 0.014 and 0.016, respectively), whereas Rb expression was lower in metastases than in primary tumours (p = 0.024). Fas receptor expression was high in normal mucosa but absent in primary tumours and metastases, whereas the opposite was seen for p53. The expression of bax, mcl-1, and bcl-x in normal mucosa was more apical than that seen in malignant cells, where a more diffuse expression pattern was seen (p < 0.04). Apoptosis was more abundant in primary tumours than in metastases. CONCLUSIONS: These results demonstrate that proliferation and apoptosis are disturbed during colorectal cancer progression, and this is accompanied by loss of Rb and Fas expression, the accumulation of p53 and TS, and changes in the expression patterns of bax, mcl-1, and bcl-xl.     

Effect of timing of oocyte denudation and micro-injection on survival, fertilization and embryo quality after intracytoplasmic sperm injection

In human in-vitro fertilization (IVF), the oocytes are surrounded by cumulus and corona cells at the time of insemination so that their maturity cannot easily be evaluated. The best IVF results are obtained if the oocytes are inseminated 2-6 h after retrieval. In the intracytoplasmic sperm injection (ICSI) procedure, the oocytes are denuded by enzymatic and mechanical treatment in order to be able to perform the injection. As a consequence, the nuclear maturity of the oocytes can be evaluated and only those that have extruded the first polar body are injected. However, metaphase-II oocytes that have not yet reached cytoplasmic maturity cannot be recognized. The purpose of this study was to investigate the effect of different timing of cumulus- corona cell removal and injection on the outcome of ICSI. For this we allowed the oocytes to complete in-vitro cytoplasmic maturation in two different culture conditions: (i) surrounded by their cumulus and corona cells or (ii) totally denuded. We performed three different studies on sibling oocytes obtained after a standardized buserelin/human menopausal gonadotrophin (HMG) protocol. We investigated the effect of early (1-2 h after retrieval) and late (5-6 h after retrieval) oocyte denudation and injection on the survival and fertilization of the injected oocytes and on embryo cleavage after fertilization. We found no statistically significant differences between early and late injection, indicating that after a standardized buserelin/HMG protocol the metaphase-II oocytes do not need time for further cytoplasmic maturation. Furthermore, a different timing of cumulus-corona cell removal has no effect on the outcome of ICSI, suggesting that the surrounding cells are not necessary for survival, fertilization and cleavage after ICSI.      

Karyotyping and DNA flow cytometry of metastatic ovarian yolk sac tumor

We karyotyped a metastasis composed of pure yolk sac tumor derived from a primary ovarian germ cell tumor with two components: a dermoid cyst [DNA index (DI) 1.0] and a pure yolk sac tumor (DI 1.88). The metastatic yolk sac tumor had a hypertriploid karyotype and a DI of 1.78 and lacked the germ cell tumor marker i(12p). The absence of this marker in a metastasis from a tumor with a dermoid cyst component might be indicative for a pathogenesis of the yolk sac tumor similar to that of a dermoid cyst and different from that of dysgerminoma.     

Expression of NAD(P)H:quinone oxidoreductase in the normal and Parkinsonian substantia nigra

Dopamine (DA) autooxidation, and consequent formation of neurotoxic DA-derived quinones and reactive oxygen species, has been implicated in dopaminergic cell death and, hence, in the pathogenesis of Parkinson's disease (PD). Stimulation of pathways involved in the detoxication of DA-quinones in the brain is hypothesized to be an effective means to limit oxidative stress and to confer neuroprotection in PD. In this respect, the inducible flavoprotein NAD(P)H:quinone oxidoreductase (NQO1) is of particular interest as it is directly implicated in the detoxication of DA-quinones and, in addition, has broad spectrum anti-oxidant properties. To study the potential pathophysiological role of NQO1 in PD, the cellular expression of NQO1 was examined in the mesencephalon of PD patients and age-matched controls. In the substantia nigra pars compacta (SNpc), NQO1 was found to be expressed in astroglial and endothelial cells and, albeit less frequently, also in dopaminergic neurons. Moreover, while overt NQO1 immunoreactivity was absent in the surrounding nervous tissue, in the Parkinsonian SNpc a marked increase in the astroglial and neuronal expression of NQO1 was consistently observed.     

Interferon-gamma (IFN-gamma) and IL-4 expressed during mercury-induced membranous nephropathy are toxic for cultured podocytes.

The subepithelial immune deposits of Dorus Zadel Black (DZB) rats with mercury-induced membranous nephropathy consist of autoantibodies directed to laminin P1 and of complement. The animals develop massive proteinuria within 10-14 days which is associated with obliteration of foot processes of glomerular visceral epithelial cells (GVEC), or podocytes. Previous studies indicate that these autoantibodies are probably not the sole mediator of proteinuria and GVEC damage. In this study we investigated whether circulating or macrophage-derived cytokines can contribute to the GVEC changes as detected in vivo. In vivo at the height of the proteinuria, increased intraglomerular IFN-gamma immunoreactivity was found. In diseased rats a five-fold increase in intraglomerular macrophages was found, but we could not detect intraglomerular IFN-alpha, IFN-beta, IL-1 beta or tumour necrosis factor-alpha (TNF-alpha) by using immunohistology. Subsequently, we exposed cultured GVEC to these cytokines to investigate their cytotoxic effects on several physiological and structural parameters. IFN-gamma and IL-4 were the only cytokines that exerted toxic effects, resulting in a rapidly decreased transepithelial resistance of confluent monolayers, which was closely associated with altered immunoreactivity of the tight junction protein ZO-1. IL-4 also affected vimentin and laminin immunoreactivity. IFN-gamma and IL-4 only interfered with monolayer integrity when added to the basolateral side of the GVEC, indicating specific (receptor-mediated) effects. Only IL-4 decreased the viability of the cells, and treated monolayers demonstrated an increased passage of the 44-kD protein horseradish peroxidase. From our experiments we concluded that IFN-gamma subtly affected monolayer integrity at the level of the tight junctions, and that IL-4 additionally induced cell death. We hypothesize that the toxic effects of the cytokines IFN-gamma and IL-4 as seen with cultured podocytes are necessary together with the autoantibodies, for the ultimate induction of proteinuria in mercury nephropathy in the DZB rat.     

Molecular epidemiology of Pseudomonas aeruginosa colonization in a burn unit: persistence of a multidrug-resistant clone and a silver sulfadiazine-resistant clone

To study the epidemiology of Pseudomonas aeruginosa colonization in a 32-bed burn wound center (BWC), 321 clinical and 45 environmental P. aeruginosa isolates were collected by prospective surveillance culture over a 1-year period and analyzed by serotyping, drug susceptibility testing, and amplified fragment length polymorphism (AFLP) analysis. Among 441 patients treated at the center, 70 (16%) were colonized with P. aeruginosa, including 12 (17%) patients who were colonized on admission and 58 (83%) patients who acquired the organism during their stay. Of the 48 distinct AFLP genotypes found, 21 were found exclusively in the environment, 15 were isolated from individual patients only, and 12 were responsible for the colonization of 57 patients, of which 2 were also isolated from the environment, but secondary to patient carriage. Polyclonal P. aeruginosa colonization with strains of two to four genotypes, often with different antibiotic susceptibility patterns, was observed in 19 patients (27%). Two predominant genotypes were responsible for recurrent outbreaks and the colonization of 42 patients (60% of all colonized patients). The strain with one of those genotypes appeared to be endemic to the BWC and developed multidrug resistance (MDR) at the end of the study period, whereas the strain with the other genotype was antibiotic susceptible but resistant to silver sulfadiazine (SSD(r)). The MDR strain was found at a higher frequency in sputum samples than the SSD(r) strain, which showed a higher prevalence in burn wound samples, suggesting that anatomic habitat selection was associated with adaptive resistance to antimicrobial drugs. Repeated and thorough surveys of the hospital environment failed to detect a primary reservoir for any of those genotypes. Cross-acquisition, resulting from insufficient compliance with infection control measures, was the major route of colonization in our BWC. In addition to the AFLP pattern and serotype, analysis of the nucleotide sequences of three (lipo)protein genes (oprI, oprL, and oprD) and the pyoverdine type revealed that all predominant strains except the SSD(r) strain belonged to recently identified clonal complexes. These successful clones are widespread in nature and therefore predominate in the patient population, in whom variants accumulate drug resistance mechanisms that allow their transmission and persistence in the BWC.