P148 CD68 expression in inflammatory cell infiltration of nonspecific invasive breast cancer

BackgroundTumor-associated macrophages play a main role in tumor progression and dissemination. Taking into account the high heterogeneicity of tumor the different clinical impact of macrophages, infiltrating different sites of tumor, could be expected. The aim was to detect the level of CD68+ cells (macrophages) in the different site of stroma in breast tumor in comparison to clinical course.Materials and methodsOne thirty-six women with nonspecific invasive breast cancer T1-4N0-3M0, who were treated in General Oncology Department of Tomsk Cancer Research Institute (Tomsk, Russia), were included in the present study. Patients did not receive preoperative treatment. The material was fixed in 10–12% neutral formalin. Preparation of the histological material was carried out according to standard procedures. Morphological examination of the surgical specimens was performed by the standard method using a light microscope “Carl Zeiss Axio Lab.A1” (Germany) and slidescanner “MiraxMidiZeiss” (Germany). Metastatic lesion was detected in regional lymph nodes. Immunohistochemical study was performed according to standard procedures. Cytoplasmic expression of these markers was determined in the inflammatory cell infiltrate of different tumor segments: (1) in areas with soft fibrous stroma; (2) in areas with coarse fibrous stroma; (3) in the areas of the so-called “maximum stromal-and-parenchymal relationship” where the individual tumor cells, short strands and groups of tumor cells arranged in soft fibrous stroma; (4) among parenchymal elements; (5) in gaps of ductal tumor structures. Double-stained immunofluorescence was performed according to standard procedures using Leica TCS SP2 laser-scanning spectral confocal microscope (Germany). The following primary antibodies were used: mouse monoclonal anti-human CD68 (BD Biosciences) and rabbit polyclonal anti-stabilin-1 or RS1 (marker of M2 macrophages).ResultsThe highest expression of CD68 in the inflammatory cell infiltrate was detected more frequently in areas with soft fibrous stroma (54%) or the so-called “maximum stromal-and-parenchymal relationship” (79%) in patients with breast cancer. The lowest expression of CD68 was observed in areas with coarse fiber stroma (23%). The CD68-positive cells of the inflammatory infiltrate were located between parenchymal elements of tumor (88%). Inverse correlation (R=−0.67; p=0.02) observed between tumor size and the expression of CD68 in the cells of the inflammatory infiltrate in gaps of tubular tumor structures. The CD68 expression in cells of the inflammatory tumor infiltrate was correlated with the presence of metastatic regional lymph nodes. It was found that in the case of the lymph node metastases the average score of CD68 expression in cells of ductal gaps tumor structures was lower (1.4±0.5) in comparison with the negative lymph nodes case (3.1±1.0; F=10.9; p=0.007). Same time no correlation between the CD68 expression in the inflammatory cell tumor infiltrate and the rate of tumor malignancy was found. Using confocal microscopy domination of CD68+/RS1+ cells were found.ConclusionSo, low CD68 expression level in ductal gaps tumor structures is associated with the presence of metastatic regional lymph nodes

Deconvoluting hepatic processing of carbon nanotubes

Single-wall carbon nanotubes present unique opportunities for drug delivery, but have not advanced into the clinic. Differential nanotube accretion and clearance from critical organs have been observed, but the mechanism not fully elucidated. The liver has a complex cellular composition that regulates a range of metabolic functions and coincidently accumulates most particulate drugs. Here we provide the unexpected details of hepatic processing of covalently functionalized nanotubes including receptor-mediated endocytosis, cellular trafficking and biliary elimination. Ammonium-functionalized fibrillar nanocarbon is found to preferentially localize in the fenestrated sinusoidal endothelium of the liver but not resident macrophages. Stabilin receptors mediate the endocytic clearance of nanotubes. Biocompatibility is evidenced by the absence of cell death and no immune cell infiltration. Towards clinical application of this platform, nanotubes were evaluated for the first time in non-human primates. The pharmacologic profile in cynomolgus monkeys is equivalent to what was reported in mice and suggests that nanotubes should behave similarly in humans

Clever-1/Stabilin-1 regulates lymphocyte migration within lymphatics and leukocyte entrance to sites of inflammation

Clever-1/Stabilin-1 is a scavenger receptor present on lymphatic and sinusoidal endothelium as well as on a subset of type II macrophages. It is also induced on vasculature at sites of inflammation. However, its in vivo function has remained practically unknown and this work addresses those unknown aspects. We demonstrate using in vivo models that Clever-1/Stabilin-1 mediates migration of T and B lymphocytes to the draining lymph nodes in vivo and identify the adhesive epitope of the Clever-1/Stabilin-1 molecule responsible for the interaction between lymphocytes and lymphatic endothelium. Moreover, we demonstrate that Ab blocking of Clever-1/Stabilin-1 efficiently inhibits peritonitis in mice by decreasing the entrance of granulocytes by 50%, while migration of monocytes and lymphocytes into the inflamed peritoneum is prevented almost completely. Despite efficient anti-inflammatory activity the Ab therapy does not dramatically dampen immune responses against the bacterial and foreign protein Ag tested and bacterial clearance. These results indicate that anti-Clever-1/Stabilin-1 treatment can target two different arms of the vasculature - traffic via lymphatics and inflamed blood vessels.</p

Analysis of Gga Null Mice Demonstrates a Non-Redundant Role for Mammalian GGA2 during Development

Numerous studies using cultured mammalian cells have shown that the three GGAs (Golgi-localized, gamma-ear containing, ADP-ribosylation factor- binding proteins) function in the transport of cargo proteins between the trans- Golgi network and endosomes. However, the in vivo role(s) of these adaptor proteins and their possible functional redundancy has not been analyzed. In this study, the genes encoding GGAs1-3 were disrupted in mice by insertional mutagenesis. Loss of GGA1 or GGA3 alone was well tolerated whereas the absence of GGA2 resulted in embryonic or neonatal lethality, depending on the genetic background of the mice. Thus, GGA2 mediates a vital function that cannot be compensated for by GGA1and/or GGA3. The combined loss of GGA1 and GGA3 also resulted in a high incidence of neonatal mortality but in this case the expression level of GGA2 may be inadequate to compensate for the loss of the other two GGAs. We conclude that the three mammalian GGAs are essential proteins that are not fully redundant

Изменение функционального профиля моноцитов крови при раке молочной железы

The purpose of the study was to identify functional features of circulation monocytes in patients with nonmetastatic breast cancer.Material and Methods. The study cohort consisted of 10 breast cancer patients treated at Tomsk Cancer Research Institute. 7 healthy female volunteers were enrolled as a control group. CD14+16-, CD14+16+ and CD14-16+ monocytes subsets were obtained from blood by sorting. Whole transcriptome profling was provided in monocytes from patients and healthy females. Macrophages were differentiated from the obtained monocytes under in vitro conditions. The ability of conditioned media obtained from macrophages to infuence apoptosis and proliferation of MDA-MB 231 cell line was evaluated.Results. Transcriptomic profling revealed signifcant changes in monocytes of breast cancer patients. CD14+16- subset showed higher expression of transporters ABCA1 and ABCG1; chemokines CCR1, CRRL2, CXCR4; maturation and differentiation factors Mafb and Jun; endocytosis mediating factors CD163 and Siglec1; proteases and tetrasponins ADAM9, CD151, CD82, and growth factor HBEGF in patient group. Macrophages derived from monocytes of breast cancer patients produced factors that supported proliferation of the MDA-MB 231 cell line, which was not observed for monocytes from healthy volunteers.Conclusion. Thus, breast carcinoma has a systemic effect on peripheral blood monocytes, programming them to differentiate into macrophages with tumor supporting capacity. Цель исследования ‒ оценить особенности функционального профиля моноцитов периферической крови у больных неметастатической формой рака молочной железы.Материал и методы. В исследование вошли 10 больных раком молочной железы II–III стадии (T1–3N0–2M0). В качестве контроля была обследована группа из 7 здоровых женщин. Моноциты были получены из периферической крови путем сортировки популяций с фенотипом CD14+16-, CD14+16+ и CD14-16+. Проведено полнотранскриптомное профилирование полученных моноцитов от больных раком молочной железы и здоровых женщин. Из полученных моноцитов in vitro были дифференцированы макрофаги. Проведена оценка способности полученных от макрофагов кондиционных сред влиять на апоптоз и пролиферацию клеток линии MDA-MB 231.Результаты. Показано, что транскриптомный профиль моноцитов больных РЖЖ имеет выраженные отличия по сравнению со здоровыми женщинами. Моноциты пациенток с раком молочной железы отличаются повышенной экспрессией мРНК белков-транспортеров ABCA1, ABCG1; хемокинов CCR1, CRRL2, CXCR4; факторов созревания и дифференцировки моноцитов Mafb и Jun; факторов, опосредующих эндоцитоз CD163, Siglec1; протеаз и тетраспонинов ADAM9, CD151, CD82 и ростового фактора HBEGF. Макрофаги, полученные в результате культивирования моноцитов больных раком молочной железы в условиях in vitro, продуцировали факторы, которые позволили поддерживать пролиферацию клеточной линии опухолевых клеток, чего не наблюдалось для моноцитов здоровых доноров.Заключение. Опухоль молочной железы оказывает системное влияние на моноциты периферической крови, программируя их к дифференцировке в макрофаги с проопухолевой функциональной активностью.

Gene Expression Patterns in Peripheral Blood Correlate with the Extent of Coronary Artery Disease

Systemic and local inflammation plays a prominent role in the pathogenesis of atherosclerotic coronary artery disease, but the relationship of whole blood gene expression changes with coronary disease remains unclear. We have investigated whether gene expression patterns in peripheral blood correlate with the severity of coronary disease and whether these patterns correlate with the extent of atherosclerosis in the vascular wall