Extremely thin layer plastification for focused-ion beam scanning electron microscopy: an improved method to study cell surfaces and organelles of cultured cells.

Since the recent boost in the usage of electron microscopy in life-science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused-ion beam scanning electron microscopy (FIB-SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold-labelled breast cancer SKBR3 cells was to visualise gold-labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 µm2 cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back-scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB-SEM. Cross-sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms.

A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy.

Immuno-electron microscopy is commonly performed with the use of antibodies. In the last decade the antibody fragment indicated as nanobody (VHH or single domain antibody) has found its way to different applications previously done with conventional antibodies. Nanobodies can be selected to bind with high affinity and specificity to different antigens. They are small (molecular weight ca. 15kDa) and are usually easy to produce in microorganisms. Here we have evaluated the feasibility of a nanobody binding to HER2 for application in immuno-electron microscopy. To obtain highest labeling efficiency combined with optimal specificity, different labeling conditions were analysed, which included nanobody concentration, fixation and blocking conditions. The obtained optimal protocol was applied for post-embedment labeling of Tokuyasu cryosections and for pre-embedment labeling of HER2 for fluorescence microscopy and both transmission and scanning electron microscopy. We show that formaldehyde fixation after incubation with the anti-HER2 nanobody, improves labeling intensity. Among all tested blocking agents the best results were obtained with a mixture of cold water fish gelatine and acetylated bovine serum albumin, which prevented a-specific interactions causing background labeling while preserving specific interactions at the same time. In conclusion, we have developed a nanobody-based protocol for immuno-gold labeling of HER2 for Tokuyasu cryosections in TEM as well as for pre-embedment gold labeling of cells for both TEM and SEM.

Bilateral ureteral obstruction due to primary myelofibrosis caused hyperuricaemia.

In healthy population, uric acid comprises the major component of 10-20% of renal stones. Extreme hiperuricaemia is seen in cancer patients with tumour lysis syndrome (TLS) which is classically associated with haematological malignancies with rapid tumour growth rates such as acute lymphoid leukaemia and high grade lymphomas. Primary melofibrosis (Agnogenic myeloid metaplasia-AMM) is a chronic myeloproliferative disease characterized by splenomegaly, a leukoerythroblastic blood picture, teardrop poikilocytosis and varying degrees of marrow fibrosis. Due to the increased extramedullary haematopoiesis, hiperuricemia may occur. However, TLS in patients with AMM is, according to the available literature, described just in one patient. In this paper we present a case of a 47-year-old male patient who was admitted to the hospital with symptoms of fatigue and small amount of urine, and clinical signs of plethora and enlarged spleen. The laboratory findings showed leuko-and erythrocytosis, increased levels of urea-BUN (32 mmol/l) and creatinine (766 mmol/l) as well as uric acid (920 mmol/l). The immediate abdominal ultrasound confirmed extreme splenomegaly, but also showed bilateral hydronephrosis of grade II-III with two stones in proximal part of right ureter and one in proximal part of left ureter as well as empty bladder. Stones were not seen on plain film. Since the patient was in complete anuria, with further rapid elevation of BUN and creatinine levels, bilateral ureteral stents were applicated together with extensive hydration, urine alkalization and administration of allopurinol which resulted in the complete recovery of kidney function. The bone marrow biopsy was also performed and histopathological diagnosis was: Hypercellulary phase of AMM.

Relationship between inflammatory cytokines and cardiorenal anemia syndrome: treatment with recombinant human erythropoietin (rhepo).

BACKGROUND AND AIM: Hemodialysis (HD) patients are exposed to persistent inflammatory state. Erythropoietin resistance is known to be strongly associated with chronic inflammation. Aim of the study was to analyze the effect of elevated inflammatory markers on hemoglobin levels and rhEPO requirements in stable patients of our hemodialysis center. PATIENTS AND METHODS: The study population consisted of 42 patients, 19F/23M, mean age 54.5+/-12.0 years. C-reactive protein (CRP), interleukin-6 (IL-6), hemoglobin (Hb), ferritin and left ventricular mass index (LVMi) were recorded. Group 1 consisted of 10 patients with Hbor=10 g/dl, mean 12.6+/-1.91 g/dl. None of these 20 patients had been previously treated with rhEPO. Group 3 consisted of 22 patients with mean Hb 10.1+/-1.5 g/dl after treatment with rhEPO. RESULTS: Group 1 patients had significantly higher IL-6 concentrations than Group 2 (5.21+/-3.94 vs 3.03+/-3.64, p<0.03). Group 3, treated with rhEPO had IL-6 concentrations significantly lower compared to Group 1 (1.15+/-3.81 vs 3.03+/-3.64, p<0.05). HD pts in Group 1 presented significantly higher CRP concentrations compated to pts of Group 2 and 3 (23.1+/-9.1 vs 7.02+/-8.7 and 7.89+/-9.6 respectively, p<0.05). A negative correlation was demonstrated between IL-6 and Hb level (r: 0.33 p<0.05). CONCLUSIONS: A better management of anemia might improve inflammatory state and survival in this population.