191. Biopsja węzła wartowniczego w operacyjnym raku gruczołu piersiowego – doświadczenia własne

Do chwili obecnej w przypadku raka piersi usunięcie układu chłonnego pachy jest obowiązkowym elementem postępowania chirurgicznego. Zajęcie węzłów chłonnych w przypadku raka piersi jest jednym z czynników rokowniczych jak i wpływa na podjęcie dalszego leczenia uzupełniającego i dlatego usunięcie pachowych węzłów chłonnych jest bardzo ważnym elementem leczenia operacyjnego raka piersi. Technika biopsji węzła wartowniczego (WW) rozwinęła się w przypadkach czerniaka złośliwego skóry i ma na celu precyzyjną ocenę stanu całego dorzecza węzłów chłonnych przy użyciu barwnika Paten Blau V, radioizotopu Tc99 ręcznej sóndy gamma kamery oraz małoinwazyjnej techniki chirurgicznej.Materiał i metodaW okresie od sierpnia 1998 roku do września 2003 roku w I Oddziale Chirurgii Onkologicznej Wielkopolskiego Centrum Onkologii w Poznaniu poddano biopsji WW 400 pacjentek z operacyjnym rakiem piersi. U wszystkich chorych klinicznie nie stwierdzano powiększonych węzłów chłonnych. Wiek pacjentek wahał się od 35 do 70 lat ze średnią wieku 55,2 lat. W przeddzień operacji podawano podskórnie w okolicę guza z czterech wkłuć Nannocoloid znaczony Tc99 w stężeniu 1 mCi zawarty w 4 mililitrach roztworu. Dnia następnego rano wykonywano limfoscyntygrafię celem wykonania mappingu węzła/węzłów wartowniczych. Następnie na sali operacyjnej podawano 1 do 2 mililitrów barwnika Patent Blau V w ten sam sposób, co radiokoloid. Przy użyciu ręcznej sondy gamma kamery identyfikowano miejsce największego wychwytu znacznika w pasze (tzw. Hot, Spot), które zaznaczano. Po odsłonięciu tkanki tłuszczowej pachy uwidaczniano wybarwione drogi chłonne, wzdłuż których dokonywano identyfikację wybarwionego węzła chłonnego. Następnie przy użyciu ręcznej sondy Navigator potwierdzano największy wychwyt promieniowania nad wybarwionym węzłem, w takim przypadku powyższy węzeł chłonny uważany był jako WW i przesyłany do pracowni patologicznej. Po identyfikacji WW chora poddawana była standardowej operacji (mastectomia lub BCT) wraz z limfadenektomią pachową jako nieodłącznym elementem procedury chirurgicznej.WynikiWW udało się zidentyfikować w 97% chorych. W przypadku 7 pacjentek nie udało się odnależć WW. Z pośród chorych z definiowanym WW przerzuty stwierdzono w 20% przypadków, w pozostałej części badanej grupy WW nie zawierał komórek nowotworowych, co stanowi 80% badanej populacji. W badanej grupie chorych stwierdzono 2 przypadki wyniku fałszywie ujemnego. W większoścr przypadków (87,1%) WW występował jako pojedynczy tylko u 2 pacjentów stwierdzono podwójny węzeł chłonny a u jednej chorej WW występował jako potrójny, ogółem mnogie WW wynosiły 2,88% populacji.WnioskiBiopsja WW jest bezpieczną metodą pozwalającą na ocenę dorzecza pachowych węzłów chłonnych w operacyjnym raku piersi. Wyniki nasze potwierdzają w pełni wyniki innych badaczy w Europie i na Świecie

54. Lymphatic mapping and sentinel lymphnode Biopsy in breast cancer patients in clinical stage T1-T2 NO MO

The authors report the feasibility an accuracy of intraoperative sentinel lymphnode biopsy (SLN) in patients with operable breast cancer to test the hypothesis that the histologic characteristic of SLN predicts the histologic status of remaining lymph nodes in the axilla. In between August 1999 and May 2000 to SLN biopsy there were 124 patients enrolled with median age of 55,2 years of age. All patients had operable breast cancer and all axillary lymphnodes were clinically negative.A day before surgery 4 cc of Nannocol marked with Tc99 was injected subcutuanesly nearby the tumor bed and an hour later the lymphoscyntygraphy was performed to achieve axillary lymphnodes mapping. Then immediately prior to surgery Blue Dye Patent Blau V was injected in the same manner as the radiological marker. Then with the use of hand – held gamma probe the hotspot was defined and marked. Five to ten minutes later first cut was performed over marked hotspot. After visualization of stained lymph vassals the SLN was traced and confirmed with high dose output (using the Neoprobe) and then was harvested for histologic examination. After this all patients were submitted for regular lymphadenectomy regardless main surgery type (mastectomy or BCT).Employing above method we were able to define SLN in 93,3%, only in 6,7% of our patients identification failed. In the group with defined SLN we found cancer cells deposits only in 20%. And in that group remaining lymphnodes had cancer metastasis in almost 80%. We had two cases of false negative results but still our specify rate was over 97%.In conclusion we think that SLN biopsy is quite safe and efficient method to evaluate axillary lymphnodes status in patients with operable early stage breast cancer. Our findings are also confirmed by other authors world wide

Distinct Levels of Sox9 Expression Mark Colon Epithelial Stem Cells that Form Colonoids in Culture

Sox9 is an high-mobility group box transcription factor that is expressed in the stem cell zone of the small intestine and colon. We have previously used a Sox9EGFP mouse model to demonstrate that discrete levels of Sox9 expression mark small intestine epithelial stem cells that form crypt/villus-like structures in a three-dimensional culture system (Formeister EJ, Sionas AL, Lorance DK, Barkley CL, Lee GH, Magness ST. Am J Physiol Gastrointest Liver Physiol 296: G1108–G1118, 2009; Gracz AD, Ramalingam S, Magness ST. Am J Physiol Gastrointest Liver Physiol 298: G590–G600, 2010). In the present study, we hypothesized that discrete levels of Sox9 expression would also mark colonic epithelial stem cells (CESCs). Using the Sox9EGFP mouse model, we show that lower levels of Sox9 mark cells in the transit-amplifying progenitor cell zone, while higher levels of Sox9 mark cells in the colonic crypt base. Furthermore, we demonstrate that variable SOX9 levels persist in cells of colonic adenomas from mice and humans. Cells expressing lower Sox9 levels demonstrate gene expression profiles consistent with more differentiated populations, and cells expressing higher Sox9 levels are consistent with less differentiated populations. When placed in culture, cells expressing the highest levels of Sox9 formed “colonoids,” which are defined as bodies of cultured colonic epithelial cells that possess multiple cryptlike structures and a pseudolumen. Cells expressing the highest levels of Sox9 also demonstrate multipotency and self-renewal in vitro, indicating functional stemness. These data suggest a dose-dependent role for Sox9 in normal CESCs and cells comprising colon tumors. Furthermore, distinct Sox9 levels represent a new biomarker to study CESC and progenitor biology in physiological and disease states

A high-throughput platform for stem cell niche co-cultures and downstream gene expression analysis

Stem cells reside in 'niches', where support cells provide critical signalling for tissue renewal. Culture methods mimic niche conditions and support the growth of stem cells in vitro. However, current functional assays preclude statistically meaningful studies of clonal stem cells, stem cell-niche interactions, and genetic analysis of single cells and their organoid progeny. Here, we describe a 'microraft array' (MRA) that facilitates high-throughput clonogenic culture and computational identification of single intestinal stem cells (ISCs) and niche cells. We use MRAs to demonstrate that Paneth cells, a known ISC niche component, enhance organoid formation in a contact-dependent manner. MRAs facilitate retrieval of early enteroids for quantitative PCR to correlate functional properties, such as enteroid morphology, with differences in gene expression. MRAs have broad applicability to assaying stem cell-niche interactions and organoid development, and serve as a high-throughput culture platform to interrogate gene expression at early stages of stem cell fate choices

Reparameterization of RNA χ Torsion Parameters for the AMBER Force Field and Comparison to NMR Spectra for Cytidine and Uridine

A reparameterization of the torsional parameters for the glycosidic dihedral angle, χ, for the AMBER99 force field in RNA nucleosides is used to provide a modified force field, AMBER99χ. Molecular dynamics simulations of cytidine, uridine, adenosine, and guanosine in aqueous solution using the AMBER99 and AMBER99χ force fields are compared with NMR results. For each nucleoside and force field, 10 individual molecular dynamics simulations of 30 ns each were run. For cytidine with AMBER99χ force field, each molecular dynamics simulation time was extended to 120 ns for convergence purposes. Nuclear magnetic resonance (NMR) spectroscopy, including one-dimensional (1D) 1H, steady-state 1D 1H nuclear Overhauser effect (NOE), and transient 1D 1H NOE, was used to determine the sugar puckering and preferred base orientation with respect to the ribose of cytidine and uridine. The AMBER99 force field overestimates the population of syn conformations of the base orientation and of C2′-endo sugar puckering of the pyrimidines, while the AMBER99χ force field’s predictions are more consistent with NMR results. Moreover, the AMBER99 force field prefers high anti conformations with glycosidic dihedral angles around 310° for the base orientation of purines. The AMBER99χ force field prefers anti conformations around 185°, which is more consistent with the quantum mechanical calculations and known 3D structures of folded ribonucleic acids (RNAs). Evidently, the AMBER99χ force field predicts the structural characteristics of ribonucleosides better than the AMBER99 force field and should improve structural and thermodynamic predictions of RNA structures

tRNA structural and functional changes induced by oxidative stress

Oxidatively damaged biomolecules impair cellular functions and contribute to the pathology of a variety of diseases. RNA is also attacked by reactive oxygen species, and oxidized RNA is increasingly recognized as an important contributor to neurodegenerative complications in humans. Recently, evidence has accumulated supporting the notion that tRNA is involved in cellular responses to various stress conditions. This review focuses on the intriguing consequences of oxidative modification of tRNA at the structural and functional level

Distinct Bradyrhizbium communities nodulate legumes native to temperate and tropical monsoon Australia

Geographic isolation and growing climate aridity played major roles in the evolution of Australian legumes. It is likely that these two factors also impacted on the evolution of their root-nodule bacteria. To investigate this issue, we applied a multilocus sequence analysis (MLSA) approach to examine Bradyrhizobium isolates originating from temperate areas of Western Australia (WA) and the tropical monsoon area of the Northern Territory (NT). The isolates were mostly collected from the nodules of legumes belonging to tribes, genera and species endemic or native to Australia. Phylogenetic analyses of sequences for the housekeeping atpD, dnaK, glnII, gyrB, recA and 16S rRNA genes and nodulation nodAgene revealed that most isolates belonged to groups that are distinct from non-Australian Bradyrhizobium isolates, which is in line with earlier studies based on 16S rRNA gene sequence analyses. Phylogenetic analysis of the nodA data allowed identification of five major Clades among the WA and NT isolates. All WA isolates grouped in a subgroup I.1 of Clade I with strains originating from temperate eastern Australia. In contrast, the NT isolates formed part of Clades I (subgroup I.2), III (subgroup III.3), IV, V and X. Of these nodA clades, Clade I, Clade IV, Clade X presumably have an Australian origin. Overall, these data demonstrate that the impact of geographic isolation of the Australian landmass is manifested by the presence of numerous unique clusters in housekeeping and nodulation gene trees. In addition, the intrinsic climate characteristics of temperate WA and tropical-monsoon NT were responsible for the formation of distinct legume communities selecting for unrelated Bradyrhizobium groups