Danuta Ostalska-Nowicka

Cytotoxicity, acute and subchronic toxicity of ionic liquid, didecyldimethylammonium saccharinate, in rats.

The aim of this study was to investigate cytotoxicity, acute and subchronic oral toxicity of an ionic liquid didecyldimethylammonium saccharinate [DDA][Sac] in rat. IC(50) values tested on six human cell lines varied from 1.44 microM to 5.47 microM. The compound tested was classified to the 4th toxicity class with a fixed LD(50) cut-off value 500 mg/kg. Organ pathology induced by [DDA][Sac] in an acute experiment included exfoliation of the surface layer of the colon and alveolar septa in lung parenchyma. In a subchronic experiment rats were administered 10, 30 and 100 mg/kg/day [DDA][Sac] for 28 days. Reduced body weight gain and slightly reduced food consumption was observed particularly in high-dose rats. Slight hematology changes were found only in mid-dose females. Statistically significant changes in clinical chemistry parameters included: increases in the ALT, SDH, ALP and GGT activities, and in glucose, blood urea nitrogen and creatinine concentrations. However, these changes did not occur in both sexes and were not dose-related with the exception of ALP in females. No treatment-related microscopic changes were observed in a subchronic experiment. Under the condition of this study the lowest-observed-adverse-effect level of [DDA][Sac] was considered to be 10 mg/kg/day.

The significance of substance P in physiological and malignant haematopoiesis.

The role of substance P (SP) in physiological haematopoiesis is well established. However, it also seems to be important in the neoplastic transformation of bone marrow, leading to the development of acute leukaemia in children, and also metastases to bone marrow of solid tumours (particularly neuroblastoma and breast cancer) in early stages of these diseases. This review summarises the available data on SP involvement in both processes. In the future, SP antagonists may be used as anti-neoplastic drugs, for example by direct or indirect blocking of tumour cell proliferation through inhibition of growth factor production and interleukin-1b synthesis.

Prognostic value of stage IV neuroblastoma metastatic immunophenotype in the bone marrow: preliminary report.

Aim: To correlate the immunophenotype of metastatic cells in the bone marrow of patients with neuroblastoma with early treatment failure. Methods: The studies were performed on bone marrow material obtained from children treated in the department of paediatric oncology, haematology, and transplantology, Poznan University of Medical Sciences, Poland from 1996 to 2003. Immunocytochemical analysis of nervous tissue markers (using the immunomax technique) was performed on 108 bone marrow preparations obtained from 36 children diagnosed with neuroblastoma (stage IV with bone marrow metastases). The analysis included expression of PGP 9.5 protein, substance P, chromogranin A, bombesin, galanin, neuropeptide Y (NPY), and vasoactive intestinal peptide in neuroblastoma metastatic cells defined by the expression of neurone specific enolase. Results: Nineteen relapses occurred within 12 months of the end of treatment. Correlation between the various markers studied and early treatment failure, using Fisher’s exact test, revealed that chromogranin A and NPY are strong indicators of an unfavourable prognosis in patients with stage IV neuroblastoma (p < 0.001 and p < 0.0002, respectively). Conclusion: Determination of metastatic cell immunophenotypes in bone marrow (particularly chromogranin A and NPY) may help establish the short term prognosis in children with neuroblastoma.

Vascular endothelial growth factor [VEGF]-C - a potent risk factor in children diagnosed with stadium 4 neuroblastoma

To evaluate the immunohistochemical expression of VEGF-C, CD34 and VEGFR-2 in cancer tissue of children diagnosed with stadium 4 neuroblastoma (NB) and correlate their presence with the survival rate of children diagnosed with that stage of the disease. Eighteen children assigned to stadium 4 composed the study group. Fourteen patients (allocated to stadium 3) formed a control group. VEGF-C, CD34 and VEGFR-2 expressions were evaluated by immunohistochemical assay. Consecutive slides incubated with anti-CD34 and anti-VEGFR-2 antibodies revealed that the two markers were colocalized within endothelial layer of the blood vessels. On the other hand, VEGF-C was expressed exclusively in tumour cells. As demonstrated by Fishers exact test, the risk of NB treatment failure (progression or relapse) as well as tumour related death, when all the patients were considered, was found to be significant in VEGF-C positive patients. VEGF-C expression in NB constitutes a potent risk factor and may direct future anti-angiogenic treatment strategy. The proximity of VEGF-C and CD34/VEGFR-2 of NB could be the equivalent of a potentially interesting VEGF-C fashion involving a tumour cell invasion into the blood vessels in an early phase of metastases promoting.

Predictive factors of late venous aortocoronary graft failure: ultrastructural studies.

Background Venous aortocoronary graft arterialization may precede a preterm occlusion in some coronary artery bypass grafting (CABG) patients. The aim of the present study was to identify ultrastructural variations in the saphenous vein wall that may have an impact on the development of venous graft disease in CABG patients. Methods The study involved 365 consecutive patients with a mean age of 62.9±9.4 years who underwent isolated CABG. The thickness and area of the whole venous wall, the tunica intima, the tunica media and the adventitia and the number and shape (length, thickness and length/thickness ratio) of the nuclei in the medial smooth muscle cells nuclei in the distal saphenous vein segments were evaluated by ultrastructural studies. Patients were followed up for 41 to 50 months (mean 45.1±5.1). Saphenous vein graft patency was assessed by follow-up coronary angiography. Logistic regression models were used to identify independent risk factors for late graft failure. Results In 71 patients significant lesions in the saphenous vein grafts were observed. The whole venous wall thickness (437.5 µm vs. 405.5 µm), tunica media thickness (257.2 µm vs. 211.5 µm), whole venous wall area (2.23 mm2 vs. 2.02 mm2) and tunica media area (1.09 mm2 vs. 0.93 mm2) were significantly larger for this group of patients than for those without graft disease. In the latter group more elongated smooth muscle cell nuclei (higher length/thickness ratio) were found in the tunica media of the saphenous vein segments. Thickening of the saphenous vein tunica media and chunky smooth muscle cell nuclei were identified as independent risk factors for graft disease development. Conclusions Saphenous vein tunica media hypertrophy (resulting in wall thickening) and chunky smooth muscle cell nuclei might predict the development of venous graft disease.

Endothelial integrity of radial artery grafts harvested by minimally invasive surgery — immunohistochemical studies of CD31 and endothelial nitric oxide synthase expressions: a randomized controlled trial

OBJECTIVE To compare the endothelial integrity of radial artery grafts harvested by minimally invasive surgery and arteries harvested conventionally for coronary artery bypass surgery (CABG) in 200 participants, who were assigned to interventions by using random allocation. METHODS An immunohistochemical procedure with monoclonal antibodies was employed to estimate CD31 antigen and endothelial nitric oxide synthase (eNOS) expressions - markers defining endothelial integrity. RESULTS The CD31 immunostaining revealed that the endothelial cell integrity of the minimally invasive harvested arteries was preserved in 76.1±7.4% of the circumference of luminal endothelium, which was similar to results obtained in conventionally harvested grafts (77.2±9.8%; not significant). On the other hand, eNOS immunostaining indicated that the endothelial integrity of the minimally invasive harvested grafts was preserved in 75.4±10.5% while in conventionally harvested grafts it was reduced to 42.4±14.5% of the total luminal endothelium circumference (P<0.05). CONCLUSIONS The endothelial integrity of radial artery grafts harvested by minimally invasive surgery is better preserved than in the grafts obtained by the conventional manner. This could play an important role in improving graft patency and might represent a preliminary condition of stable functioning in coronary arterial bypasses.

Ezrin—a useful factor in the prognosis of nephrotic syndrome in children: an immunohistochemical approach

Background: Minimal change disease (MCD) and diffuse mesangial proliferation (DMP) are the most common pathomorphological forms of nephrotic syndrome glomerulopathies in children. The clinical course of DMP can be characterised by either DMP-sensitivity (DMP-S) or DMP-resistance (DMP-R) to steroids, resulting in an unfavourable course of the glomerulopathy. Although the clinical processes of DMP-S and DMP-R are initially identical, resistance to steroids may be foreseen by the immunohistochemical expression of cytoskeleton-associated proteins in podocytes. Aims: To estimate the immunohistochemical expression of ezrin in children with MCD, DMP and focal segmental glomerulosclerosis (FSGS) and to evaluate its usefulness in predicting resistance to steroids. Materials and methods: Renal biopsy specimens of patients with MCD (n = 15), DMP (n = 16) and FSGS (n = 6) were taken. The control tissue consisted of normal-appearing cortex taken from kidneys resected for localised neoplasms (n = 6). The indirect immunohistochemical protocol for the use of a monoclonal antibody directed against ezrin was used. Results: The immunohistochemical expression of ezrin in cases progressively reduced from MCD to DMP-S to DMP-R to FSGS. Except for DMP-R and FSGS (p>0.05), the difference in ezrin expression in podocytes was significant. Conclusion: Ezrin can be a potent marker of podocyte injury (podocytopathy) and may help in the histological qualification of MCD, DMP and FSGS. The increased permeability of the filtration barrier in steroid-resistant and proteinuric glomerulopathies may be a consequence of subcellular changes in podocyte-associated proteins following decreased expression of ezrin.

Retrospective cohort study of familial hypomagnesaemia with hypercalciuria and nephrocalcinosis due to CLDN16 mutations

BACKGROUND Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare autosomal recessive tubular disorder exhibiting a high risk for progressive chronic kidney disease (CKD). METHODS This is a retrospective multicentre study of 25 paediatric cases with FHHNC in Poland. Median age at diagnosis was 4 years and median follow-up time was 4.8 years. RESULTS All cases of FHHNC carried recessive mutations in CLDN16. The founder mutation in CLDN16, Leu151Phe, was the most frequent cause of FHHNC in Polish patients, with 13 (52%) cases being homozygous and 5 (20%) carrying Leu151Phe allele in compound heterozygosity. All cases showed nephrocalcinosis, increased urinary fractional excretion of magnesium and hypercalciuria. Other disease features included hypomagnesaemia (76%), hyperparathyroidism (76%), hyperuricaemia (56%) and hypocitraturia (60%). Treatment with thiazides effectively reduced hypercalciuria in most cases. During follow-up, renal function declined in 60% of patients; 12% of patients reached CKD stage 3 or 4 and one patient developed end-stage renal failure. CONCLUSIONS We report one of the largest cohorts of FHHNC cases caused by CLDN16 mutations. A missense variant of CLDN16, Leu151Phe, is the most common mutation responsible for FHHNC in Poland. Additionally, we found that normomagnesaemia does not exclude FHHNC and the calculation of fractional excretion of Mg can be diagnostic in the setting of normomagnesaemia. We also demonstrate the efficacy of a treatment with thiazides in terms of hypercalciuria in the majority of patients.

Preexisting High Expression of Matrix Metalloproteinase-2 in Tunica Media of Saphenous Vein Conduits Is Associated with Unfavorable Long-Term Outcomes after Coronary Artery Bypass Grafting

Introduction. Migration of the smooth muscle cells (SMCs) to the tunica media in the saphenous vein (SV) transplants is facilitated by matrix metalloproteinases (MMPs). The aim of this study was to identify any associations between expression of MMP-2 or endogenous tissue inhibitors (TIMP-2 and TIMP-3) in the SV segments and late failure of the SV grafts. Methods. Two hundred consecutive patients with a mean age of 63.1 ± 8.9 years who underwent primary isolated venous CABG were examined. Patients were retrospectively split into two subgroups, with the SV graft disease (SVGD (+); n = 47) or without it (SVGD (−); n = 153). In the SV segments, immunohistochemical analysis of the expression of the MMP-2, TIMP-2, and -3 was performed. Results. In the SVGD (+) patients, tissue expression of MMP-2 was stronger, whereas that of both TIMPs was weaker than in the SVGD (−) patients. In majority of the SV segments obtained from the SVGD (−) individuals, a balance in MMP and TIMP expressions was found, whereas an upregulation of MMP-2 expression was usually noted in the SVGD (+) subjects. Conclusion. The strong expression of MMP-2 accompanied by reduced immunostaining of both TIMPs is associated with the development of the SV graft disease and unfavorable CABG outcomes.

Histological evaluation of age-related variations in saphenous vein grafts used for coronary artery bypass grafting.

Introduction Venous coronary artery bypass grafts (CABG) might undergo a process of arterialization resulting in neointimal formation and medial hypertrophy. It is often followed by critical occlusion of the graft lumen. The aim of the study was to assess histological representative features of saphenous vein reconstruction in aging as well as to establish optimal patients’ age limits applicable for optimal selection of grafts. Material and methods One hundred and ten patients undergoing venous CABG were divided into 4 age subgroups: (A) 50 years and less, (B) 51-60 years, (C) 61-70 years and (D) > 70 year-old subjects. Distal venous graft segments were saved for an adequate morphometric assay which was followed by suitable statistical analysis. Results The entire venous wall thickness as well as its tunica media were found to become significantly thinner between subgroups A and D. The number of smooth muscle cell (SMC) nuclei within the tunica media did not differ between study subgroups. The majority of these nuclei in subgroup D were found, however, to be more elongated than in subgroup A (SMC length/width index in subgroup D was found to be significantly higher than in subgroup A). Conclusions Progressive, age-related thinning of the venous wall and tunica media as well as SMC nucleus elongation might suggest impairment of SMCs’ migration and proliferation rate. Consequently, individuals aged 70 years and over may benefit clinically more from venous CABG than younger patients due to the lower risk of arterialization and occlusion of the graft lumen in the future.