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Quddus, Iram, Farhana Samir, Humera Waqar, and Sarwer Qureshi. "Concomitant Use Of L-arginine With High Butter And Corn Oil Diet Prevent Their Harmful Effects On Adrenocortical Cells Of Albino Rats." Journal of Bahria University Medical and Dental College 08, no. 01 (December 5, 2017): 35–39. http://dx.doi.org/10.51985/jbumdc2018009.
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Objective: To compare the bichemical and morphological effects of L -Arginine against the changes caused by butter and corn oil supplementation Study design: A prospective experimental study Place: Department of Anatomy BMSI, JPMC Duration: August to October 2008. Methodology:Male Albino rats weighing 200 - 240gm were selected and divided into 5 groups. Group ‘CL’ received standard laboratory diet. Group ‘Bu’ received 20% added unsalted butter in diet. Group ‘Co’ received 20% added corn oil in diet. Group ‘BuAr’ received 20% Butter with L-Arginine 300mg /kg body weight /day orally .Group‘CoAr’ received 20%corn oil along with L-Arginine 300mg/kg body weight/day orally. On completion of study period that is 4 weeks, animals were sacrifised. Blood was drawn for hormonal assays. Adrenal glands were removed and fixed in buffered neutral formalin. Right adrenals were processed and sectioned at4 µm thickness to be stained with Mallory trichrome stain to visualize blood vessel. Left adrenalswere sectioned with cryostat in 10µm sections and stained with Oil red O to visualize fat in cells. Results:Highly significant and moderately significant decrease observed in ACTH (Adrenocorticotrophic hormone)levels in Group BuArand CoAr when compared to Bu and Co respectively; insignificant difference was found between BuAr&CoAr. Moderately significant and significant decrease observed in corticosterone levels in Group BuAr and CoAr when compared to Buand Co respectively. Insignificant difference was found between BuAr and CoAr . Mallory trichrome stained section showed less dilated blood vessels in BuAr&CoAr compared to Bu & Co respectively, while difference among the former two was not remarkable. Oil red O stained sections showed less densely packed fat globules in group BuAr&CoAr compared to Bu and Co respectively. Difference between BuAr&CoAr was not marked. Conclusion: Butter has more stimulatory effect on adrenal cortical cells but the comparison with corn oil is not statistically significant except for ACTH levels. L Arginine seems to be effective in lowering the levels of stress hormones, fat accumulation and vasodilatation when given along with corn oil and butter oil.
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Monge, Florencia A., Adeline M. Fanni, Patrick L. Donabedian, Jonathan Hulse, Nicole M. Maphis, Shanya Jiang, Tia N. Donaldson, et al. "Selective In Vitro and Ex Vivo Staining of Brain Neurofibrillary Tangles and Amyloid Plaques by Novel Ethylene Ethynylene-Based Optical Sensors." Biosensors 13, no. 2 (January 18, 2023): 151. http://dx.doi.org/10.3390/bios13020151.
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The identification of protein aggregates as biomarkers for neurodegeneration is an area of interest for disease diagnosis and treatment development. In this work, we present novel super luminescent conjugated polyelectrolyte molecules as ex vivo sensors for tau-paired helical filaments (PHFs) and amyloid-β (Aβ) plaques. We evaluated the use of two oligo-p-phenylene ethynylenes (OPEs), anionic OPE12- and cationic OPE24+, as stains for fibrillar protein pathology in brain sections of transgenic mouse (rTg4510) and rat (TgF344-AD) models of Alzheimer’s disease (AD) tauopathy, and post-mortem brain sections from human frontotemporal dementia (FTD). OPE12- displayed selectivity for PHFs in fluorimetry assays and strong staining of neurofibrillary tangles (NFTs) in mouse and human brain tissue sections, while OPE24+ stained both NFTs and Aβ plaques. Both OPEs stained the brain sections with limited background or non-specific staining. This novel family of sensors outperformed the gold-standard dye Thioflavin T in sensing capacities and co-stained with conventional phosphorylated tau (AT180) and Aβ (4G8) antibodies. As the OPEs readily bind protein amyloids in vitro and ex vivo, they are selective and rapid tools for identifying proteopathic inclusions relevant to AD. Such OPEs can be useful in understanding pathogenesis and in creating in vivo diagnostically relevant detection tools for neurodegenerative diseases.
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Tantisatirapong, Suchada, and Wongsakorn Preedanan. "Texture Based Classification of Malaria Parasites from Giemsa-Stained Thin Blood Films." ECTI Transactions on Electrical Engineering, Electronics, and Communications 18, no. 1 (February 28, 2020): 9–16. http://dx.doi.org/10.37936/ecti-eec.2020181.208115.
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Quantification of parasitemia is an important part of a microscopic malaria diagnosis. Giemsa-stained thin blood smear is the gold standard method for detecting malaria parasite enumeration. However, manual counting reveals the limitations of human inconsistency and fatigue, as well as the unreliability of accuracy and non-reproducibility. Inaccurate parasitemia affects clinical diagnosis and therapeutic procedure. Automated quantification is therefore useful to improve the performance of quantifying parasite density. In this paper, the texture-based classification approach is investigated. The methods consist of the following processes: pre-processing, segmentation, feature extraction and the classification of erythrocytes. The pre-processing is applied for image conversion and enhancement. The segmentation combines local adaptive thresholding, morphological process and watershed transform to extract red blood cells, separate touching and overlapping cells. Texture analysis is performed to establish parameters obtained from first-order, second-order and higher-order statistical analysis and wavelet transform. Two feature selection approaches, the sequential forward selection method and sequential backward elimination method, integrated with a support vector machine classifier are examined to obtain the optimal feature set for identifying the Plasmodium falciparum stages. We found that gray-level co-occurrence matrices based textural features were highly selected. The optimal feature set selected by the sequential forward selection yields lesser number of features and tends to give a higher degree of accuracy than the feature set selected by sequential backward elimination. The proposed method produces 98.87% accuracy for binary classification, 99.56% accuracy for ring stage classification, and 99.48% accuracy for tropozoite stage classification. Grey-level co-occurrence matrices based texture analysis is the dominant method compared to first-order and higher-order statistical texture analysis as well as wavelet transform.
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Hwang, Kevin, Grace Vezeau, Edyta Olejnik, Douglas Wood, Ruben Cardenes, Lauren Duro, Gourab Chatterjee, and Je H. Lee. "Abstract 3767: A novel method to minimize HIER-induced alterations on H&E staining in an integrated mIF-H&E workflow." Cancer Research 84, no. 6_Supplement (March 22, 2024): 3767. http://dx.doi.org/10.1158/1538-7445.am2024-3767.
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Abstract Background: Hematoxylin and eosin staining (H&E) is widely used as an anatomical assay for clinical diagnosis. Researchers and clinicians also rely on molecular in situ techniques, such as multiplexed immunofluorescence (mIF), to gain deeper insights on cellular phenotypes and tissue microenvironment. As a result, there has been significant interest in combining anatomical stains with molecular imaging techniques, most commonly by using a terminal H&E stain after mIF staining. This allows a combination of the two assays but has been observed to show alterations in the H&E staining pattern. In this work we identify heat-induced epitope retrieval (HIER) as the root cause of alterations of the terminal H&E stain after mIF. We further demonstrate a new workflow combining an initial H&E stain with subsequent InSituPlex assay, to avoid these alterations and thus enable co-registration of highly sensitive multiplexed immunofluorescence with an unaltered H&E stain. Methods: FFPE tissue slides were stained using a standard HIER protocol or a full mIF assay, followed by a standard H&E stain. Slides were imaged using a Zeiss Axioscan.Z1 scanner. Additional H&E-stained slides were prepared, and the H&E stain removed using a destaining protocol before carrying out InSituPlex® mIF staining for multiple markers. Fluorescence and brightfield images of the same slide were overlaid using UltiStacker® to assess qualitative differences between pre- and post-mIF H&E, and UltiAnalyzer.AI® was used to quantify cell densities and signal intensities between mIF image pre- and post-H&E. Results: Alterations in H&E staining were observed between slides directly stained with H&E and slides stained with a terminal H&E after HIER alone or a full mIF assay. Calculated cell counts and tissue area were consistent throughout, but some tissue microstructures could be difficult to resolve after either HIER or full mIF. Our destaining protocol was successful in removing hematoxylin and eosin from directly-stained H&E slides and allowing subsequent mIF staining. For most biomarkers, qualitative and quantitative differences in InSituPlex mIF staining were minimal. Brightfield and fluorescent images were co-registered with sub-micron accuracy using UltiStacker, enabling molecularly defined cellular phenotyping within a traditional H&E image. Conclusions: While the mIF to H&E workflow is widely used and is capable of allowing combined anatomical and cellular phenotyping, alterations in the H&E stain exist due to the use of HIER in most mIF protocols. An alternative method is possible in which H&E-stained slides are destained and then restained for mIF or other spatial assays, providing the same valuable combination of assay information but without the HIER-induced alterations in H&E staining pattern. Citation Format: Kevin Hwang, Grace Vezeau, Edyta Olejnik, Douglas Wood, Ruben Cardenes, Lauren Duro, Gourab Chatterjee, Je H. Lee. A novel method to minimize HIER-induced alterations on H&E staining in an integrated mIF-H&E workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3767.
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Alatrash, Gheath, Pariya Sukhumalchandra, Mao Zhang, Celine Kerros, Anna Sergeeva, Amanda Cernosek, Haroon Jakher, et al. "PR1 Is Cross-Presented By Multiple Myeloma Cells in Patients and Renders Multiple Myeloma Susceptible to PR1-CTL and Anti-PR1/HLA-A2 Antibody." Blood 124, no. 21 (December 6, 2014): 2133. http://dx.doi.org/10.1182/blood.v124.21.2133.2133.
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Abstract PR1 is an HLA-A2-retricted, nonameric peptide that is derived from the azurophil granule proteases neutrophil elastase (NE) and proteinase 3 (P3). PR1 has been targeted successfully in acute (AML) and chronic (CML) myeloid leukemia using anti-PR1/HLA-A2 antibody (8F4), PR1-peptide vaccine and PR1-specific cytotoxic T lymphocytes (PR1-CTL). We have previously reported that NE and P3 are cross-presented by normal B cells and dendritic cells (DC), leading to PR1 expression by HLA-A2. Since multiple myeloma (MM) is a B cell malignancy, we investigated whether MM cells can cross-present PR1 as a possible target for immunotherapy. To study whether PR1 is presented by MM cells, patient bone marrow (BM) was stained with 8F4 antibody and then imaged using confocal microscopy. PR1/HLA-A2 was detected on the surface of CD138+ MM cells in BM samples from 3 of 6 HLA-A2+ MM patients (Fig. 1). We then investigated whether cellular immunity to PR1 is detected in peripheral blood (PB) from patients with MM. PB samples from MM patients who had undergone allogeneic (allo) (n=9) and autologous (auto) (n=2) stem cell transplantation (SCT) were stained with PR1/HLA-A2 dextramer in addition to standard lineage markers. PR1-specific CD8+CTL were detected in PB of 10 of 11 patients (range=0.02%-2.9%). Because P3 and NE expression is limited to myeloid cells, we sought to determine the mechanism of PR1 presentation in MM. We performed RT-PCR and western blotting on seven MM cell lines, including U266, ARK, ARP-1, OPM-2, LP-1, IM-9 and RPMI 8226. Neither NE nor P3 were detected in the MM cell lines studied at either the mRNA or proteins levels. We then investigated whether MM cells took up NE and P3. We cultured MM cells for 30 hours with 10 ug/mL of soluble NE and P3 or irradiated HLA-A2 negative PMN, the latter as a source for cell-associated NE and P3. Cells were then stained intracellularly for NE and P3 at different time points. Flow cytometry analysis showed that all the cell lines analyzed took up NE and P3. Uptake was seen as early as 1 hour after co-culture with soluble NE and P3 and was higher for soluble P3. Additionally, more uptake was seen in the cells that were co-cultured with irradiated PMN in comparison with soluble NE and P3. We then investigated whether PR1 expression in MM was through NE and P3 cross-presentation. We focused our studies on the HLA-A2+ U266 MM cell line. U266 cells were co-cultured with soluble NE, P3 or irradiated PMN, as described in the previous section, and then surface stained with 8F4. We detected PR1/HLA-A2 on the surface of U266 cells by flow cytometry as early as 6 and 24 hours after co-culture with soluble NE and P3, respectively. Cross-presentation was also seen in the cells that were co-cultured with irradiated PMN to a similar extent in comparison with the cells that were cultured with soluble NE and P3, however, cross-presentation occurred at an earlier time point (1 hour) in the cells that were cultured with PMNs. Cross-presentation was abrogated by lactacystin, a proteasome inhibitor, and brefeldin A, an ER/Golgi transport inhibitor, indicating that NE and P3 cross-presentation occurs through conventional cross-presentation mechanisms. Furthermore, because immune modulatory drugs and proteasome inhibitors are part of the standard of care therapy for MM, and since both have been shown to affect cross-presentation by DC, we tested whether lenalidomide and bortezomib altered PR1 cross-presentation by U266 and normal DC. U266 and DC were cultured with irradiated PMN in the presence of increasing doses of bortezomib or lenalidomide and then stained with PR1/HLA-A2. Flow-cytometry analysis showed a significant inhibition of PR1-cross-presentation by U266 after addition of bortezomib, but not lenalidomide. PR1 cross-presentation by DC was not affected by either drug. Finally, we tested whether PR1 cross-presentation caused MM cells to become susceptible to PR1-targeting immunotherapies. U266 cells were cultured with NE or P3 for 24 hours, the time point that showed the maximal cross-presentation, followed by addition of 8F4 antibody or co-culture with PR1-CTL. Using calcein AM cytotoxicity assays, we showed dose dependent killing of U266 by 8F4 and PR1-CTL following PR1 cross-presentation (Fig. 2). Together our data show that PR1 is cross-presented by primary MM cells and cell lines. These findings lay the foundation for the future applications of PR1-targeting immunotherapies in MM. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Nikolskiy, I., V. Nikolskaya, D. Demchenko, and D. Zubov. "Potentiation of directed osteogenic differentiation of thymic multipotent stromal cells by prior co-cultivation with thymocytes." Cell and Organ Transplantology 4, no. 2 (November 30, 2016): 220–23. http://dx.doi.org/10.22494/cot.v4i2.59.
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It is known that multipotent stromal cells (MSCs) and thymocytes possess membrane affinity and interaction in the thymic niches that is essentially important for thymocytes differentiation. However there are no data about possible influence of intercellular contacts in the reverse direction: from the thymocytes to the MSCs.Materials and methods. The MSCs were obtained from the thymuses of С57ВL mice, using the explants technique, and cultivated under standard conditions during 8-12 passages. Thymocytes or bone marrow cells (106) were added to 4×104 MSCs for 24 hours. Thereafter they were eliminated and standard culture medium was changed by osteogenic or adipogenic differentiation medium and cultured during 10 days. After fixation the cells were stained by 1 % alizarin red S solution or 0.2 % solution of oil red О respectively. After extraction of the stains with 10 % acetic acid or isopropyl alcohol the optic density of extracts at 520 nm was measured.Results. We found that thymic multipotent stromal cells of the C57BL mice were effectively differentiated in vitro into the osteogenic and adipogenic lineages in the appropriate differentiation media that was evidenced by alizarin red and oil red staining of cell cultures. According to the results of measurement of optic density of the dye extracts, it was found that effectiveness of thymic MSCs differentiation into the osteogenic lineage after prior short-term co-cultivation with the thymocytes is increased.Conclusions. The contact of thymic stromal cells with thymocytes but not with bone marrow cells in the previous 24 hours potentiates the osteogenic differentiation and has no effect on the adipogenic cells maturation.
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Aertker, Benjamin M., Akshita Kumar, Fanni Cardenas, Franciska Gudenkauf, David Sequeira, Alan R. Prossin, Amit K. Srivastava, Charles S. Cox, and Supinder S. Bedi. "PET Imaging of Peripheral Benzodiazepine Receptor Standard Uptake Value Increases After Controlled Cortical Impact, a Rodent Model of Traumatic Brain Injury." ASN Neuro 13 (January 2021): 175909142110141. http://dx.doi.org/10.1177/17590914211014135.
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Traumatic brain injury (TBI) is a chronic, life threatening injury for which few effective interventions are available. Evidence in animal models suggests un-checked immune activation may contribute to the pathophysiology. Changes in regional density of active brain microglia can be quantified in vivo with positron emission topography (PET) with the relatively selective radiotracer, peripheral benzodiazepine receptor 28 (11 C-PBR28). Phenotypic assessment (activated vs resting) can subsequently be assessed (ex vivo) using morphological techniques. To elucidate the mechanistic contribution of immune cells in due to TBI, we employed a hybrid approach involving both in vivo (11 C-PBR28 PET) and ex vivo (morphology) to elucidate the role of immune cells in a controlled cortical impact (CCI), a rodent model for TBI. Density of activated brain microglia/macrophages was quantified 120 hours after injury using the standardized uptake value (SUV) approach. Ex vivo morphological analysis from specific brain regions using IBA-1 antibodies differentiated ramified (resting) from amoeboid (activated) immune cells. Additional immunostaining of PBRs facilitated co-localization of PBRs with IBA-1 staining to further validate PET data. Injured animals displayed greater PBR28suv when compared to sham animals. Immunohistochemistry demonstrated elevated density of amoeboid microglia/macrophages in the ipsilateral dentate gyrus, corpus callosum, thalami and injury penumbra of injured animals compared to sham animals. PBR co-stained with amoeboid microglia/macrophages in the injury penumbra and not with astrocytes. These data suggest the technologies evaluated may serve as bio-signatures of neuroinflammation following severe brain injury in small animals, potentially enabling in vivo tracking of neuroinflammation following TBI and cellular-based therapies.
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DOONABOYINA, RAGHAVA, Abhilasha Mittal, and Sridhar Babu Gummadi. "IN VITRO SULFORHODAMINE B ASSAY EVALUATION OF NOVEL 2-PHENYL BENZOFURANONE DERIVATIVES ON HUMAN SKIN CANCER CELL LINE G361." Journal of Drug Delivery and Therapeutics 9, no. 4-A (August 30, 2019): 385–89. http://dx.doi.org/10.22270/jddt.v9i4-a.3434.
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The newly synthesized compounds are being tested for in vitro anticancer activity. The method used for In Vitro testing is Sulforhodamine B assay also known SRB assay. Cell lines were prepared and homogenized and disassociated with the help of trypsin. Then trypsin was inactivated with fetal bovine serum. Then cell concentration was determined. Synthesized molecules were prepared into four different dilutions and exposed to cell lines. The procedure was also compared with standard drug doxorubicin. All the cell medium were incubated 37 degrees centigrade in a humidified incubator with 5 percentage CO. The plates were stained and fixed with trichloroacetic acid. Finally, the plates were incubated in orbital shaker incubator and absorbance was measured in a microplate reader at 510nm. All compounds (1-30) showed the similar anticancer activity of compounds (IA, IB, ID, IE, IF, IIB, IIC, IIIA, IVB, IVF, VA, VC, VD, VE.) were more potent when compared to the rest of the compounds synthesized.
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Pantic, Igor, Sanja Dacic, Predrag Brkic, Irena Lavrnja, Senka Pantic, Tomislav Jovanovic, and Sanja Pekovic. "Application of Fractal and Grey Level Co-Occurrence Matrix Analysis in Evaluation of Brain Corpus Callosum and Cingulum Architecture." Microscopy and Microanalysis 20, no. 5 (June 26, 2014): 1373–81. http://dx.doi.org/10.1017/s1431927614012811.
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AbstractThis aim of this study was to assess the discriminatory value of fractal and grey level co-occurrence matrix (GLCM) analysis methods in standard microscopy analysis of two histologically similar brain white mass regions that have different nerve fiber orientation. A total of 160 digital micrographs of thionine-stained rat brain white mass were acquired using a Pro-MicroScan DEM-200 instrument. Eighty micrographs from the anterior corpus callosum and eighty from the anterior cingulum areas of the brain were analyzed. The micrographs were evaluated using the National Institutes of Health ImageJ software and its plugins. For each micrograph, seven parameters were calculated: angular second moment, inverse difference moment, GLCM contrast, GLCM correlation, GLCM variance, fractal dimension, and lacunarity. Using the Receiver operating characteristic analysis, the highest discriminatory value was determined for inverse difference moment (IDM) (area under the receiver operating characteristic (ROC) curve equaled 0.925, and for the criterion IDM≤0.610 the sensitivity and specificity were 82.5 and 87.5%, respectively). Most of the other parameters also showed good sensitivity and specificity. The results indicate that GLCM and fractal analysis methods, when applied together in brain histology analysis, are highly capable of discriminating white mass structures that have different axonal orientation.
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Szabolcs, M. J., A. Windisch, R. Koller, and M. Pensch. "Axon typing of rat muscle nerves using a double staining procedure for cholinesterase and carbonic anhydrase." Journal of Histochemistry & Cytochemistry 39, no. 12 (December 1991): 1617–25. http://dx.doi.org/10.1177/39.12.1719070.
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We developed a method for detecting activity of axonal cholinesterase (CE) and carbonic anhydrase (CA)--markers for motor and sensory nerve fibers (NFs)--in the same histological section. To reach this goal, cross-sections of muscle nerves were sequentially incubated with the standard protocols for CE and CA histochemistry. A modified incubation medium was used for CA in which Co++ is replaced by Ni++. This avoids interference of the two histochemical reactions because Co++ binds unspecifically to the brown copper-ferroferricyanide complex representing CE activity, whereas Ni++ does not. Cross-sections of the trapezius muscle nerve containing efferent and afferent NFs in segregated fascicles showed that CE activity was confined to motor NFs. Axonal CA was detected solely in sensory NFs. The number of labeled motor and sensory NFs determined in serial cross-sections stained with either the new or the conventional technique was not significantly different. Morphometric analysis revealed that small unreactive NFs (diameter less than 5 microns) are afferent, medium-sized ones (5 microns less than d less than 7 microns) are unclassifiable, and large ones (d greater than 7 microns) are efferent. The heterogenous CE activity of thick (alpha) motor NFs is linked to the type of their motor units. "Fast" motor units contain CE reactive NFs; "slow" ones have CE negative neurites.
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Kobayashi, Hirofumi, Michael E. Kallen, Raymond Kozikowski, Kevin de Haan, Zihang Fang, Serge Alexanian, L. a. V. Carvalho, and Yair Rivenson. "Real-time, multiplexed rendering of lymphoma diagnostic panels from unstained tissue sections using virtual staining." JCO Global Oncology 9, Supplement_1 (August 2023): 115. http://dx.doi.org/10.1200/go.2023.9.supplement_1.115.
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115 Background: Lymphoproliferative disorders are complex, heterogeneous diseases with an ever expanding array of subtypes and variants requiring stepwise assay approach to arrive at a precise diagnosis. While extensive characterization of lesional cells and the microenvironment (TME) via immunophenotyping and genotyping remains the gold standard, a full workup may be unachievable due to limited input tissue, turnaround time pressure, or unavailability of particular markers in-house. Even when available, the interpretation of large numbers of markers by pathologists is limited by the ability to mentally spatially align markers by recall-superimposition of single-plex markers, limiting the discovery of patterns relevant for novel treatments strategies. Here we present a deep-learning based virtual staining method which transforms a single unstained tissue section into a panel of perfectly aligned virtual immunohistochemical (IHC) stained images. Methods: 4um unstained sections were cut from 30 formalin-fixed paraffin-embedded blocks. Using a standard commercial slide scanner (Axio Scan.Z1, Zeiss), autofluorescence images were captured from unstained sections and paired with brightfield images captured from the same sections after chemical staining with H&E or various IHC stains of interest. The virtual staining was performed by a deep neural network trained as a conditional GAN using accurately co-registered patches of paired images . The training dataset comprised seven types of lymphoma plus benign lymphoid tissues. Results: We successfully created models for virtual CD3, CD8, CD20 and CD30 IHC stains as well as H&E. Analysis by board certified pathologists confirmed consistent and accurate results between virtual and IHC stained slides in a range of lymphomas and reactive lymphoid tissue. We quantitatively evaluated the co-expression of CD3/CD8 and the spatial distribution of all four markers in eight holdout slides. We demonstrated that our virtual multiplex IHC staining achieves comparable performance for WSI scoring and cellular localization with actual staining across lymphoma types. Conclusions: We present the feasibility of spatially aligned, rapid virtual H&E and IHC staining of four different markers from single, label-free autofluorescence images using deep-learning techniques. To the best of our knowledge, this is the first report of a panel of virtual IHC stains, perfectly aligned and with overlaid H&E structural context, for analysis of neoplastic and reactive lymphoid tissues. Our technique provides an opportunity for a rapid, comprehensive histopathology and immunophenotyping workflow. Multiplex virtual staining greatly expands diagnostic and discovery opportunities: high accuracy TME profiling, seamless downstream computational image analysis, and screening of large biomarker pools in retrospective analysis of clinical trials.
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Naderi, Misagh, Nikola Mitic, Nikola Spasic, Djordje Cikic, Kristen Ruf, Nikolaus Wagner, Igor Mihajlovic, et al. "Abstract 7391: Beyond traditional staining: A virtual-IHC digital assay for HER2 score prediction from routine H&E images." Cancer Research 84, no. 6_Supplement (March 22, 2024): 7391. http://dx.doi.org/10.1158/1538-7445.am2024-7391.
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Abstract Background: In breast cancer diagnostics, the accurate assessment of HER2 status is essential for treatment stratification. Conventionally, HER2 immunohistochemistry (IHC) scoring necessitates specialized staining protocols, which are resource- and time-intensive. This study presents a virtual IHC (vIHC) digital assay to predict HER2 scores directly from hematoxylin and eosin (H&E) stained images, which are routinely acquired for all specimens. This methodology aims to accelerate standard practices by reducing the need for additional IHC staining, thereby decreasing laboratory workload, costs, and turnaround time. It also presents an opportunity to screen a wider array of samples promptly, potentially identifying candidates for HER2-targeted therapy that might otherwise be delayed or overlooked. Methods: Serially sectioned breast core needle biopsy samples were stained with H&E and HER2-IHC following Leica-Bond staining protocols. Stained slides were imaged using the Hologic GeniusTM Digital Diagnostics System with Research Use Only software for Whole Slide Imaging. From this initial image dataset, 240 regions of interest (ROIs) were manually selected in order to create a dataset that uniformly covers: (i) all HER2-IHC scores (0-3), (ii) a wide range of HER2 stain intensities, and (iii) expression patterns. The IHC scores were assigned to each selected ROI by a pathology expert, following the scoring recommendations of ASCO/CAP American Society of Clinical Oncology and College of American Pathologists, with the caveat that ductal carcinoma in situ was also given a score for the purpose of training the model. These ROIs were then extracted from the images of the serially sectioned H&E-stained slides using Reveal Bio’s proprietary co-registration digital assay. A predictive digital assay was developed at Reveal Biosciences to classify the 240 H&E-stained breast tissue ROIs into four HER2 IHC score categories: 0, 1, 2, and 3. For model evaluation, cross-validation was performed 10 times by randomly splitting the dataset of H&E ROIs into 80% for training MIL and 20% for testing the model, followed by averaging the performance metrics across the 10 dataset splits. The performance metrics included classification accuracy, precision, recall, and F1 scores for each HER2 score category. Results: The vIHC digital assay for predicting the HER2 scores directly from H&E stained breast tissue images achieved the average classification accuracy of 93% across the four HER2 score categories on the test set of ROIs. This performance demonstrates that the model is capable of successfully distinguishing between the HER2 score categories directly from H&E images without the need for IHC staining. The results suggest that the vIHC digital assay could be a reliable and cost-effective tool for preliminary breast cancer screening, providing a valuable adjunct to traditional histopathological analysis. Citation Format: Misagh Naderi, Nikola Mitic, Nikola Spasic, Djordje Cikic, Kristen Ruf, Nikolaus Wagner, Igor Mihajlovic, Sinisa Todorovic, Ray Jenoski, Sid Mayer, Michael Quick, Claire Weston. Beyond traditional staining: A virtual-IHC digital assay for HER2 score prediction from routine H&E images [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7391.
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Travain, Willian, Hanna Carolina Bet Dos Santos, Valéria Schoffen Romao Carrascoza, Márcia Do Nascimento Brito, and Célia Regina Godoy Gomes. "Efeito do Óleo de Coco sobre a Morfologia da Aorta de Ratos Obesos." Saúde e Pesquisa 8, no. 1 (June 22, 2015): 35. http://dx.doi.org/10.17765/1983-1870.2015v8n1p35-43.
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O objetivo desse estudo foi avaliar a influência do óleo de coco sobre morfologia da aorta torácica. Foram utilizados 23 animais, com 120 dias de idade. Os animais foram inicialmente separados em dois grupos, controle (tratado com dieta padrão para roedores) e obeso (tratado com dieta hipercalórica para induzir a obesidade). Aos 90 dias, os animais dos grupos controle e obeso foram subdivididos em dois grupos adicionais: C/ag, C/co, O/ag, O/co. Aos 120 dias foram eutanasiados com pentobarbitol sódico. As aortas torácicas extraídas foram processadas histologicamente e coradas com Tricrômico de Masson e Orceína-picrussírus-hematoxilina para visualização dos componentes colágeno, músculo liso e elástico, seguida da quantificação realizada por densidade de volume. Realizou-se a medida íntima-média, para verificação da espessura do vaso. A estatística seguiu Análise de Variância de um fator (ANOVA), seguida pelo teste de Tukey, para nível de significância de 5%. Concluímos que a utilização de óleo de coco em ratos obesos parece induzir uma remodelação vascular marcada pela diminuição na composição de colágeno e aumento de células musculares lisas em ratos obesos tratados com óleo de coco. Effects of Coconut Oil on the Morphology of Obese Rats´ Aorta ABSTRACT: Current study analyzes the influence of coconut oil on the morphology of the thorax aorta in 23 rats aged 120 days. The animals were separated into two groups, control (treated with standard ration for rodents) and obese (treated with a hypercaloric diet to induce obesity). After 90 days the control and obese groups were subdivided into two additional groups: C/ag, C/co, O/ag, O/co. On the 120th day the rats were euthanized with sodium pentobarbitol. The thorax aortas were removed and histologically processed. They were stained with Masson Trichromium and orcein-picrosirius-hematoxylin to visualize collagen components, smooth and elastic muscle, followed by volume density. Vessel thickness was evaluated by intima-media measure. Analysis of variance (ANOVA) and Tukey´s test were employed at 5% significance. The use of coconut oil in obese rats may have induced vascular re-modeling by decreasing the composition of collagen and increasing the smooth muscle cells in obese rats treated with coconut oil.
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Pokorska-Bocci, Anna, Sandrine Micallef, Mariola Dymkowska, Yuval Gabay, Roman Gluskin, Avi Laniado, Efrat Dicker Sagy, et al. "Predicting response to naratuximab emtansine, an anti-CD37 antibody-drug conjugate (ADC), in combination with rituximab in diffuse large B-cell lymphoma (DLBCL), by analyzing the spatial arrangement of CD37 and CD20 positive cells using deep learning." Journal of Clinical Oncology 40, no. 16_suppl (June 1, 2022): e19565-e19565. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.e19565.
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e19565 Background: DLBCL is the most common type of Non-Hodgkin’s lymphoma, accounting for 30-40% of cases. Despite improvements in response and survival with standard of care treatment, up to 40% of patients have relapsed and/or refractory (R/R) disease. A phase 2 study (NCT02564744) evaluated the efficacy of naratuximab emtansine, an anti-CD37 ADC, in combination with rituximab, in 80 patients with R/R DLBCL. We performed an exploratory analysis of the study to find pathology-based biomarkers predictive of response. Deep learning (DL) models were used to extract spatial features from digitized slides stained with CD37 and CD20 and their predictive role was evaluated. Methods: A cohort of 47 DLBCL patients from the study were selected based on availability of CD20/CD37 IHC staining and were used for analysis. Patient characteristics of the analyzed cohort were similar to those of the full study cohort. Overall response rate (ORR) of the analyzed cohort was 44.7%, similar to the ORR of the full study cohort. For each patient, whole slide images (WSI) of two slides from a pre-treatment biopsy, one stained for CD20 and one for CD37, were analyzed. DL models were used to classify cells as positive or negative to the two markers and CD20/CD37 co-expression was analyzed. Over 140 spatial features were pre-defined based on drug mechanism of action and biological hypotheses, and were calculated for each patient using cell classifications. A repeated 5-fold cross-validation analysis was performed to identify features predictive of objective response. Results: Two spatial features related to the proximity of CD37 and CD20 positive cells, demonstrated a significant correlation with clinical outcome. Each feature identified patients in the cohort as having either a positive or a negative response. Feature performance was estimated using 2000 repetitions of 5-fold cross-validation. One feature classified on average 21% of patients as responders. Patients positive for this feature had an average ORR of 78% (95% CI 64%-82%), an increase of 34% in ORR relative to the original cohort (p < 0.05). The second feature classified on average 35% of patients as positive. Patients positive for this feature had an average ORR of 67% (95% CI 62%-71%), an increase of 23% in ORR relative to the original cohort (p < 0.05). Conclusions: DL analysis of the co-expression and spatial arrangement of CD37 and CD20 in pre-treatment biopsies of DLBCL patients could potentially be used as a predictive biomarker for a response to a combination treatment of anti-CD37 and anti-CD20 drugs in DLBCL, and may improve patient stratification for further clinical trials.
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Khan, AR, M. Cocker, JD Spence, M. Alturkustani, C. Currie, C. Cathie, L. Hammond, et al. "3D carotid reconstructions: imaging, pathology, algorithms and pipelines." Canadian Journal of Neurological Sciences / Journal Canadien des Sciences Neurologiques 42, S1 (May 2015): S37. http://dx.doi.org/10.1017/cjn.2015.170.
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Background: Whole-slide scanning of tissue sections spatially informed by imaging studies offers the opportunity to reconstruct specimens for co-registration to 3D imaging data. Digital image analysis algorithms can be designed to analyze and reconstruct such specimens via electronic “pipelines”. Methods: A goal of the Canadian Atherosclerosis Imaging Network (CAIN) is to improve the assessment of carotid atheromatous disease through studies that inform clinical imaging with gold-standard data (plaque pathology). To achieve this, sectioned atheromas are manually annotated and analyzed by electronic algorithm for pathological features of interest. Resulting images are then reassembled in 3D for registration to ultrasound, CT, PET-CT and MRI studies. Results: Carotid endarterectomy specimens were sub-serially sectioned, stained, digitized and annotated manually and by electronic algorithms. Resulting 2D images were successfully rendered, reassembled and analyzed in 3D using ex-vivo micro-CT as a spatial reference. Furthermore, histology quantification using colour deconvolution was found to be preferred over hue-saturation-intensity methods 94.7-100% of the time in a blinded multiple rater study. Conclusion: Automated “pipelines” greatly facilitate 3D reconstruction in comparison to traditional slice-by-slice methods. Transformations spatially guided by pre-existing imaging data is not only faster, but has superior objectivity and fidelity. With embedded annotations, 3D pathology maps become a rich, micron-level, permanent digital pathological database for correlative studies.
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Jovanovic-Medojevic, Milica, Vanja Opacic-Galic, Katarina Geler, and Dusan Pavlica. "Comparison of the efficiency of cleaning and disinfection protocols for hand endodontic instruments." Vojnosanitetski pregled, no. 00 (2022): 103. http://dx.doi.org/10.2298/vsp220913103j.
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Background/Aim. There is no standard protocol for cleaning and desinfection of used endodontic instruments before their sterilization and reuse. The aim of this study wss to check the efficiency of the different methods of removing biological debris from different type of used hand stainless steel endodontic instruments. Methods. A total of 120 instruments (forty Kerr-Reamers, Kerr-Files and Hedstr?m-Files) (Shenzhen Denco Medical Co., Ltd. Guangdong, China) ISO 25 were analyzed. The instruments used for root canal treatment on extracted teeth were divided into 4 groups based on different decontamination protocols. The evaluation of the efficiency of the cleaning methods was based on the evaluation of the amount of stained organic residues (Van Gieson staining). Samples were analyzed by stereomicroscopy (X40). Statistical analysis was performed by Mann-Whitney and ANOVAOneWay/Bonfferoni test at a significance level of 5% (? = 0.05). Results. Residual biological debris was observed on 93.33% of all the samples taken. The thermal disinfectant cleaning method showed the lowest contamination values for all types of instruments. The method of mechanical cleaning showed the mean value of Maximum Biologic Contamination (MBC) of the Kerr-Reamers (58.54 %) and Kerr-Files (56.25%) and for Hedstr?m-Files the highest MBC was shown by the ultrasonic method contamination (50.21 %). Conclusion. The use of a thermal disinfectant was the most efficient cleaning method for all three types of hand endodontic instruments.
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Meah, Arafat, Vadanasundari Vedarethinam, Robert Bronstein, Nehaben Gujarati, Tanya Jain, Sandeep K. Mallipattu, Yueming Li, and Jun Wang. "Single-Cell Spatial MIST for Versatile, Scalable Detection of Protein Markers." Biosensors 13, no. 9 (August 27, 2023): 852. http://dx.doi.org/10.3390/bios13090852.
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High-multiplex detection of protein biomarkers across tissue regions has been an attractive spatial biology approach due to significant advantages over traditional immunohistochemistry (IHC) methods. Different from most methods, spatial multiplex in situ tagging (MIST) transfers the spatial protein expression information to an ultrahigh-density, large-scale MIST array. This technique has been optimized to reach single-cell resolution by adoption of smaller array units and 30% 8-arm PEG polymer as transfer medium. Tissue cell nuclei stained with lamin B have been clearly visualized on the MIST arrays and are colocalized with detection of nine mouse brain markers. Pseudocells defined at 10 μm in size have been used to fully profile tissue regions including cells and the intercellular space. We showcased the versatility of our technology by successfully detecting 20 marker proteins in kidney samples with the addition of five minutes atop the duration of standard immunohistochemistry protocols. Spatial MIST is amenable to iterative staining and detection on the same tissue samples. When 25 proteins were co-detected on 1 mouse brain section for each round and 5 rounds were executed, an ultrahigh multiplexity of 125 proteins was obtained for each pseudocell. With its unique abilities, this single-cell spatial MIST technology has the potential to become an important method in advanced diagnosis of complex diseases.
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Rittel, Miriam F., Stefan Schmidt, Cleo-Aron Weis, Emrullah Birgin, Björn van Marwick, Matthias Rädle, Steffen J. Diehl, Nuh N. Rahbari, Alexander Marx, and Carsten Hopf. "Spatial Omics Imaging of Fresh-Frozen Tissue and Routine FFPE Histopathology of a Single Cancer Needle Core Biopsy: A Freezing Device and Multimodal Workflow." Cancers 15, no. 10 (May 10, 2023): 2676. http://dx.doi.org/10.3390/cancers15102676.
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The complex molecular alterations that underlie cancer pathophysiology are studied in depth with omics methods using bulk tissue extracts. For spatially resolved tissue diagnostics using needle biopsy cores, however, histopathological analysis using stained FFPE tissue and the immunohistochemistry (IHC) of a few marker proteins is currently the main clinical focus. Today, spatial omics imaging using MSI or IRI is an emerging diagnostic technology for the identification and classification of various cancer types. However, to conserve tissue-specific metabolomic states, fast, reliable, and precise methods for the preparation of fresh-frozen (FF) tissue sections are crucial. Such methods are often incompatible with clinical practice, since spatial metabolomics and the routine histopathology of needle biopsies currently require two biopsies for FF and FFPE sampling, respectively. Therefore, we developed a device and corresponding laboratory and computational workflows for the multimodal spatial omics analysis of fresh-frozen, longitudinally sectioned needle biopsies to accompany standard FFPE histopathology of the same biopsy core. As a proof-of-concept, we analyzed surgical human liver cancer specimens using IRI and MSI with precise co-registration and, following FFPE processing, by sequential clinical pathology analysis of the same biopsy core. This workflow allowed for a spatial comparison between different spectral profiles and alterations in tissue histology, as well as a direct comparison for histological diagnosis without the need for an extra biopsy.
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Lee, S. H., E. M. N. Setyawan, and B. C. Lee. "166 Paracrine Regulation of Epidermal Growth Factor-Like Factors in Oviduct Cells and its Effect on Oocyte Maturation and Subsequent Embryo Development." Reproduction, Fertility and Development 30, no. 1 (2018): 222. http://dx.doi.org/10.1071/rdv30n1ab166.
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Progesterone (P4) and progesterone receptor signalling appears essential for maintenance of a proper cumulus cell expansion during the oocyte maturation by regulating the epidermal growth factor-like factors (EGF-F) related pathway during the ovulatory process. It is known that expression of EGF-F including amphiregulin (AREG), epiregulin (EREG), and betacellulin (BTC) is critical for cumulus–oocyte complex (COC) expansion and resumption of meiosis. Therefore, we hypothesised that oviduct cells might be involved in nonexclusive mechanisms of actions of P4 that in turn modulate oocyte meiosis resumption by regulating the levels of EGF-F. First, we added different concentrations of P4 (0, 0.5, 1, and 2 μg mL−1) to oviduct cell culture medium and assessed the effect of P4 on expression of AREG, EREG, and BTC in oviduct cells by immunocytochemical analysis. Then, the oviduct cells were used for co-culturing under the proper concentration of P4 with porcine oocytes. The COC were randomly cultured in 3 groups: (1) culturing without oviduct cells, (2) co-culturing with oviduct cells, and (3) co-culturing with oviduct cells treated with P4. After IVM, extrusion of the 1st polar body was observed under the microscope. To evaluate embryo development competence, the matured oocytes were activated with electrical stimulus and parthenotes were cultured in porcine zygote medium-5 for 7 days at 39°C, 5% CO2 and O2 in a humidified atmosphere. The cleavage and blastocyst formation rates were observed under the microscope to evaluate developmental competence. To count the total cell number of blastocysts, they were stained with 5 μg mL−1 of Hoechst 33342 for 10 min. The data were analysed by one-way ANOVA using GraphPad Prism 5.0 (GraphPad Inc., San Diego, CA, USA). Values are means ± standard error of mean (P < 0.05). Significantly higher levels of EGF-F were observed in oviduct cells treated with 1 μg mL−1 progesterone. The oocyte maturation rate of co-culture group treated with P4 (80.7 ± 1.6%) was significantly higher than that of the control (69.7 ± 2.1%). There was a significant difference between co-culture treated with P4 and the control in cleavage rate (67.2 ± 2.4% and 82.0 ± 1.6%). However, no significant difference was observed between the co-culture groups. The co-culture treated with P4 group showed significantly higher rate of blastocyst formation (37.7 ± 0.8%) and total cell number of blastocyst (72.8 ± 1.0) than control and co-culture groups. In conclusion, co-culturing with oviduct cell treated with P4 improved oocyte maturation and subsequent embryo development. Thus, we suggested that oviduct cells induce the expression of EGF-F under the treatment of P4, which has a beneficial effect on porcine oocyte development. This research was supported by NRF-20142A1021187, Korea IPET (#316002-05-2-SB010), RDA (#PJ010928032017) and Research Institute for Veterinary Science, the BK21 plus program.
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Li, Pengfei, Hongli Luo, Yanhui Chen, Yating Wang, Huan Chen, Lihong Wu, Yegang Ma, and Henghui Zhang. "Immune biomarker expression in the tumor microenvironment in Chinese patients with esophageal squamous cell carcinoma was explored." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e15542-e15542. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e15542.
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e15542 Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies in China, of which the standard treatment of advanced esophageal cancer is chemotherapy followed by surgery. Recently, immunotherapy has shown great potential in ESCC treatment. The aim of this study was to explore the landscape of immune-related gene expression in the tumor microenvironment (TIME) and further explore the relationship between immune biomarkers and therapeutic effect. Methods: We retrospectively analyzed PDL1(clone sp142)/PD1 expression in a cohort of Chinese patients (male = 92, female = 18; median age = 64, range from 36 to 84) with ESCC. Patient samples were detected in Genecast. Co. from February 2017 to November 2018. Another 6 patients (male = 4, female = 2) who accepted anti-PD1/PD-L1 antibody treatment were analyzed. PD-L1/PD-1 expression and the incidence of CD8, CD57, CD68, CD163 TILs were immunohistochemically. Results: In tumor cells, more than 32% (36/110) samples of ESCC patients was stained with PDL1 (≥1%), while52.8% (21/36) of these patients highly expressed PDL1 (≥10%, 10%-98%). In tumor infiltrating lymphocytes (TILs), 31.82% (35/110) patients expressed PDL1 (≥1%) and 1.82% (2/110) highly expressed PDL1 (≥10%, 10%-50%). For PD1 expression in TILs, 17.27% (19/110) samples were positively stained with PD1 antibody (≥1%). In a subset of ESCC patients who received immune checkpoint blockade therapy (including 2 responders and 4 non-responders), the median percentage of CD163, CD68, PDL1, CD8, PD1 and CD57 were 1.49% (0.44%-9.98%), 0.40% (0.16%-19.08%), 3.74% (0%-45%), 9.52% (4.20%-13.79%), 4.51% (1.37%-33.43%) and 0.68% (0.08%-1.74%), respectively in tumor area and 4.14% (1.85%-15.99%), 1.62% (0.21%-11.47%), 4.87% (0.33%-18.97%), 7.74% (5.88%-14.35%), 3.93% (1.92%-20.54%) and 0.92% (0.27%-2.18%), respectively in stroma area. When compared with non-responders, samples from the responders showed higher CD8 expression in tumor area. Conclusions: Percentage of PDL1/PD1 expression is relatively high in Chinese patients with ESCC. High levels of CD8 positive TILs might be beneficial to patients with ESCC. Clinical investigations with large sample sizes are warranted to prove our findings.
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Bhattarai, Shristi, Geetanjali Saini, Hongxiao Li, Gaurav Seth, Timothy B. Fisher, Emiel A. M. Janssen, Umay Kiraz, Jun Kong, and Ritu Aneja. "Predicting Neoadjuvant Treatment Response in Triple-Negative Breast Cancer Using Machine Learning." Diagnostics 14, no. 1 (December 28, 2023): 74. http://dx.doi.org/10.3390/diagnostics14010074.
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Background: Neoadjuvant chemotherapy (NAC) is the standard treatment for early-stage triple negative breast cancer (TNBC). The primary endpoint of NAC is a pathological complete response (pCR). NAC results in pCR in only 30–40% of TNBC patients. Tumor-infiltrating lymphocytes (TILs), Ki67 and phosphohistone H3 (pH3) are a few known biomarkers to predict NAC response. Currently, systematic evaluation of the combined value of these biomarkers in predicting NAC response is lacking. In this study, the predictive value of markers derived from H&E and IHC stained biopsy tissue was comprehensively evaluated using a supervised machine learning (ML)-based approach. Identifying predictive biomarkers could help guide therapeutic decisions by enabling precise stratification of TNBC patients into responders and partial or non-responders. Methods: Serial sections from core needle biopsies (n = 76) were stained with H&E and immunohistochemically for the Ki67 and pH3 markers, followed by whole-slide image (WSI) generation. The serial section stains in H&E stain, Ki67 and pH3 markers formed WSI triplets for each patient. The resulting WSI triplets were co-registered with H&E WSIs serving as the reference. Separate mask region-based CNN (MRCNN) models were trained with annotated H&E, Ki67 and pH3 images for detecting tumor cells, stromal and intratumoral TILs (sTILs and tTILs), Ki67+, and pH3+ cells. Top image patches with a high density of cells of interest were identified as hotspots. Best classifiers for NAC response prediction were identified by training multiple ML models and evaluating their performance by accuracy, area under curve, and confusion matrix analyses. Results: Highest prediction accuracy was achieved when hotspot regions were identified by tTIL counts and each hotspot was represented by measures of tTILs, sTILs, tumor cells, Ki67+, and pH3+ features. Regardless of the hotspot selection metric, a complementary use of multiple histological features (tTILs, sTILs) and molecular biomarkers (Ki67 and pH3) resulted in top ranked performance at the patient level. Conclusions: Overall, our results emphasize that prediction models for NAC response should be based on biomarkers in combination rather than in isolation. Our study provides compelling evidence to support the use of ML-based models to predict NAC response in patients with TNBC.
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Yeo, L., N. Adlard, M. Biehl, M. Juarez, T. Smallie, M. Snow, C. D. Buckley, K. Raza, A. Filer, and D. Scheel-Toellner. "Expression of chemokines CXCL4 and CXCL7 by synovial macrophages defines an early stage of rheumatoid arthritis." Annals of the Rheumatic Diseases 75, no. 4 (April 9, 2015): 763–71. http://dx.doi.org/10.1136/annrheumdis-2014-206921.
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Background and objectivesFor our understanding of the pathogenesis of rheumatoid arthritis (RA), it is important to elucidate the mechanisms underlying early stages of synovitis. Here, synovial cytokine production was investigated in patients with very early arthritis.MethodsSynovial biopsies were obtained from patients with at least one clinically swollen joint within 12 weeks of symptom onset. At an 18-month follow-up visit, patients who went on to develop RA, or whose arthritis spontaneously resolved, were identified. Biopsies were also obtained from patients with RA with longer symptom duration (>12 weeks) and individuals with no clinically apparent inflammation. Synovial mRNA expression of 117 cytokines was quantified using PCR techniques and analysed using standard and novel methods of data analysis. Synovial tissue sections were stained for CXCL4, CXCL7, CD41, CD68 and von Willebrand factor.ResultsA machine learning approach identified expression of mRNA for CXCL4 and CXCL7 as potentially important in the classification of early RA versus resolving arthritis. mRNA levels for these chemokines were significantly elevated in patients with early RA compared with uninflamed controls. Significantly increased CXCL4 and CXCL7 protein expression was observed in patients with early RA compared with those with resolving arthritis or longer established disease. CXCL4 and CXCL7 co-localised with blood vessels, platelets and CD68+macrophages. Extravascular CXCL7 expression was significantly higher in patients with very early RA compared with longer duration RA or resolving arthritisConclusionsTaken together, these observations suggest a transient increase in synovial CXCL4 and CXCL7 levels in early RA.
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Sefland, Oystein, Reidun Kristin Kopperud, Hester Van Zeeburg, Jeroen Rovers, Maria Omsland, Arjan A. Van de Loosdrecht, and Bjorn T. Gjertsen. "Intradermal Vaccination with Vididencel in MRD+ AML-Patients Leads to Increase in Antigen Presenting Cells and T-Cells to the Injection Site, Visualized Using Imaging Mass Cytometry, Showing Local Immune Cell Interactions Leading to Systemic Immune Responses." Blood 142, Supplement 1 (November 28, 2023): 2942. http://dx.doi.org/10.1182/blood-2023-180813.
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Background. Vididencel is an allogeneic leukemia-derived dendritic cell vaccine being developed as maintenance treatment in AML patients. Intradermal vaccination using vididencel is known to induce a local skin reaction, with local antigen processing by skin dendritic cells and direct presentation to T cells as well as dendritic cells, crawling out of the skin and presentation of the antigen in the draining lymph nodes for a full-blown immune response. It was previously shown that vididencel is able to induce an influx of immune cells in the skin using standard immunohistochemistry (van de Loosdrecht, 2018). In a phase 2 study vididencel was investigated as AML maintenance treatment after induction of CR1 with chemotherapy (ADVANCE-II, Clintrials.gov: NCT03697707). Using imaging mass cytometry up to 43 markers can be assessed in the same tissue section, to allow in depth analysis of the immune cells on a single cell level infiltrating in the dermis at the injection site. Methods. AML patients, in first complete remission with measurable residual disease (MRD +) and not planned for HSCT at inclusion, received 4 biweekly doses of vididencel followed by 2 booster doses at week 14 and 18. Skin biopsies of the injection site were taken 2 days after the 1 st, 4 th and 6 th vaccination. Control samples were collected from unvaccinated/unaffected skin in two patients and from healthy controls. Samples were fixed and embedded in paraffin, and stained by standard immunohistochemistry for immune cell markers like CD4, CD8 and CD68 in the central lab. For patients treated at Haukeland University Hospital (N=6), tissue sections were prepared, mounted and stained with the panel of 29 antibodies labelled with heavy metal isotopes for use in the Hyperion mass cytometry imaging. For each sample a region of interest (ROI) of 2.8mm 2 was scanned based on the localization of the immune infiltrates, guided by standard H&E staining from neighboring slides. Results In 88.5% of patients injection site reactions were reported in this phase 2 study, characterized by redness, swelling and warmth at the site of intradermal injection, which were mild (maximum grade 1 or 2), and were of short duration (generally resolved within 12 days). Skin biopsies of a subset of 6 patients were analyzed using Hyperion. In standard single stain immunohistochemistry a large influx of CD4, CD8 and CD68 cells was observed in contrast to normal skin. Imaging mass cytometry confirmed these dense immune infiltrations. Using additional markers to characterize monocytes, dendritic cells and T-cells revealed that these cells are in close proximity and suggestive for cell-to-cell interaction (Figure 1). At sites of dense immune infiltration, influx of GranzymeB expressing CD8 T-cells and CD38 positive CD4 T-cells were observed as well as CD1c+ or CD141+ dendritic cells with high HLA-DR expression. These T-cells and dendritic cells are in close contact suggestive for patients' dendritic cells migrating from blood to skin, inducing a local immune response. Conclusion/discussion. Vaccination with vididencel triggered an inflammatory skin reaction in the majority of patients, with redness, warmth and swelling, of short duration, indicative for an active immune response at the site of injection. Skin biopsies using standard immunohistochemistry showed influx of activated CD4 and CD8 T cells as well as antigen presenting cell, including dendritic cells.Extended analysis with multiparameter imaging mass cytometry showed specific immune cell subsets infiltrating in these site of injection sites. The observed co-localization of the different immune subsets following vaccination supports the hypothesis of local antigen capturing by blood derived and local antigen presenting cells (Zuo et al, 2021). The local immune response may ultimately leads to a systemic immune response and disease control.
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Sartain, Sarah E., Nancy A. Turner, Hui Shiu-Ki, Charles G. Minard, and Joel L. Moake. "Factor H (FH) Is Released Slowly and Continuously from Human Endothelial Cells (ECs): FH Is Not Packaged in Weibel-Palade Bodies or Secreted in Response to EC Stimulation." Blood 124, no. 21 (December 6, 2014): 4175. http://dx.doi.org/10.1182/blood.v124.21.4175.4175.
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Abstract Introduction Ultra-large von Willebrand factor (ULVWF) strings are synthesized in ECs, packaged in Weibel-Palade Bodies (WPBs), and secreted by stimulated ECs. Complement components studied to date [C3, factor (F) B, FD, FP, FH, FI, C5] are released slowly and continuously from human umbilical vein endothelial cell (HUVEC) cytoplasm and are not packaged in WPBs (PLoS One. 2013;8(3):e59372). In contrast, a recent report (Blood. 2014;123(1):121-5) contended that FH co-localizes with VWF in the WPBs. If this were so, it could have therapeutic importance for the treatment of atypical hemolytic uremic syndrome (aHUS) resulting from deficiency of FH because it might be possible to increase circulating FH levels transiently by administration of the WPB secretagogue, des-amino-D-arginine vasopressin (DDAVP). Hypothesis FH is not co-localized with VWF in WBPs, but rather is released slowly and continuously from EC cytoplasm regardless of cell stimulation. Methods Immunofluorescent Microscopy HUVECs were stimulated with histamine and stained with rabbit anti-VWF plus secondary donkey anti-rabbit Alexa Fluor IgG-488. The cells were then fixed and stained with goat-anti FH plus secondary chicken anti-goat Alexa Fluor IgG-647. The nuclei were detected with DAPI. In vitro VWF and FH from HUVECs HUVECs either were, or were not, stimulated with histamine. Supernatant was collected a variety of times over 7 hrs and assayed for VWF and FH antigen levels by ELISA. VWF assay antibodies (polyclonal): (1) capture, rabbit anti-human VWF (Ramco); (2) detection, goat anti-human VWF (Bethyl) and rabbit anti-goat IgG-HRP (Invitrogen). FH assay antibodies: (1) capture, polyclonal goat anti-human FH (Advanced Research Technologies); (2) detection, monoclonal mouse anti-human FH (Pierce, Thermo Scientific) and polyclonal goat anti-mouse IgG-HRP (Invitrogen). In vivo VWF and FH Plasma samples were obtained from 6 pediatric patients with von Willebrand disease (VWD) being tested for EC release of WPB VWF in response to DDAVP. For each patient, 1 sample was obtained prior to DDAVP administration, and 2 other samples were obtained 1 and 4 hours later. VWF levels were measured in each sample using standard clinical laboratory procedure at an affiliated hospital. FH antigen levels were quantified by ELISA, as above. Results Using non-overlapping spectral secondary detection antibody pairs, VWF was seen in clusters in HUVEC WPBs (Fig. 1A). In contrast, FH was distributed throughout the HUVEC cytoplasm (Fig. 1B). The VWF and FH images did not overlap, indicating that VWF and FH did not co-localize in the WPBs. Fig 1. Immunofluorescent images of HUVECs stained for VWF and FH. Fig 1. Immunofluorescent images of HUVECs stained for VWF and FH. Histamine addition to HUVECs in vitro resulted in ~ 4-fold increases in VWF secreted from HUVEC WPBs at 30 min and 1 hour, and 2-fold increases at 3 hours (Fig 2A). In contrast, FH release was slow and continuous, regardless of histamine stimulation, suggesting that FH is located in EC cytoplasm and is not stored in WPBs (Fig. 2B). Fig 2. In vitro VWF and FH release from ECs under non-stimulated and histamine-stimulated conditions. Fig 2. In vitro VWF and FH release from ECs under non-stimulated and histamine-stimulated conditions. In all 6 patient samples, VWF antigen increased significantly at 1-hour post-DDAVP administration (Fig. 3A). In contrast, FH antigen levels did not change significantly at hour 1 or hour 4, compared to hour 0, indicating that FH is not co-localized and secreted along with VWF from the WPBs of stimulated ECs in vivo (Fig. 3B). Fig 3. (A) Mean responses of VWF antigen to DDAVP, in vivo, with 95% CIs.The mean response was significantly greater 1-hour and 4-hours post-DDAVP compared with baseline (P=0.0085 and 0.0079, respectively). After 1-hour post-DDAVP, the mean response was 201% greater (95% CI: 129%, 314%) than baseline. After 4-hours, the mean response was 174% greater (95% CI: 123%, 247%) than baseline. (B) Responses of FH to DDAVP, in vivo. There was no statistically significant difference in FH response between time points (P=0.77). Fig 3. (A) Mean responses of VWF antigen to DDAVP, in vivo, with 95% CIs.The mean response was significantly greater 1-hour and 4-hours post-DDAVP compared with baseline (P=0.0085 and 0.0079, respectively). After 1-hour post-DDAVP, the mean response was 201% greater (95% CI: 129%, 314%) than baseline. After 4-hours, the mean response was 174% greater (95% CI: 123%, 247%) than baseline. (B) Responses of FH to DDAVP, in vivo. There was no statistically significant difference in FH response between time points (P=0.77). Conclusions We used immunofluorescent microscopy and ELISA assays on samples obtained in vitro and in vivo to demonstrate that FH is not packaged in, or secreted from, the WPBs of stimulated human ECs. FH is, therefore, similar to all other complement components studied to date in that it is released slowly and continuously from ECs and is not influenced by cell stimulation. DDAVP is unlikely to be a viable treatment option for patients with aHUS secondary to deficiency or inhibition of FH. Disclosures Sartain: Hemostasis and Thrombosis Research Society: Research Funding. Turner:Mary R Gibson Foundation: Research Funding; Hinkson Memorial Fund : Research Funding. Moake:Mary R Gibson Foundation: Research Funding; Hinkson Memorial Fund: Research Funding.
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Takahashi, Hiromichi, Sumiko Kobayashi, Katsuhiro Miura, Daisuke Kurita, Yoshihiro Hatta, Masahiko Sugitani, Shimon Otake, et al. "Clinical Significance of Co-Expression of MYC and BCL2 Protein in Advanced Diffuse Large B-Cell Lymphoma Treated with a Dose-Intensified Immunochemotherapy." Blood 126, no. 23 (December 3, 2015): 3940. http://dx.doi.org/10.1182/blood.v126.23.3940.3940.
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Abstract Background Recent studies have shown that the concurrent expression of MYC and BCL2 protein evaluated by immunohistochemistry (IHC) in patients with de novo diffuse large B-cell lymphoma (DLBCL) is associated with worse survival when treated with standard R-CHOP, but the effect of intensive chemotherapies for such patients is unknown. Thus, we evaluated the impact of the co-expression of MYC and BCL2 protein among patients with advanced DLBCL, who were treated with a dose-intensive immunochemotherapy followed by up-front autologous stem cell transplantation (ASCT). Patients and Methods This is a retrospective analysis of patients with de novo DLBCL, who were categorized into high/high-intermediate risk by the age-adjusted International Prognostic Index (aaIPI). They were consecutively treated with the R-Double-CHOP regimen, consisting of rituximab (375 mg/m2, day -2), cyclophosphamide (750 mg/m2, day 1, 2), doxorubicin (50 mg/m2, day 1, 2), vincristine (1.4 mg/m2 [maximum 2.0 mg/body], day 1) and prednisolone (50 mg/m2, day 1-5) followed by consolidative high-dose chemotherapies at our institution from 2001 to 2013. MYC and BCL2 protein were measured by IHC assay using formalin-fixed paraffin-embedded tissue specimens for all available cases. Cut-off values of positivity for MYC and BCL2 protein were set as 40% and 50% of stained tumor cell, respectively. Lymphomas showing concurrent positivity for MYC and BCL2 protein were defined as "Double expressor lymphoma (DEL)". Results A total of 40 patients with a median 53-years (range 19-68) of age were analyzed. Twenty-one patients were at high risk and the other 19 patients were at high-intermediate risk by aaIPI. Cell of origin (COO) subtypes classified by Hans algorithm consisted of 14 germinal center B-cell (GCB) type and 26 non-GCB type. Totally, 10 (25%) patients were categorized into DEL. The overall response (OR) and the complete response (CR) rates to R-Double-CHOP for all patients were 93% and 83%, respectively. The OR and the CR rates were not significantly different between the DEL group and the non-DEL group (100% vs 90%, and 80% vs 83%, respectively). The proportion of patients proceeding to ASCT was not significantly different among these groups (50% vs 60%). With a median 52 months (range 3-155) of follow-up, the 3-year progression-free survival (PFS) and the overall survival (OS) rates for all patients were 55% and 72%, respectively (Figure a, b). Both the PFS and the OS were significantly worse in the DEL group than in the non-DEL group (Figure c, d). As for aaIPI and COO subtyping, either high/high-intermediate risk or GCB/non-GCB subtype were not significantly associated with the outcome of PFS or OS. Conclusion The concurrent expression of MYC/BCL2 protein in advanced DLBCL was associated with shorter remission duration and worse survival despite similar susceptibility to the treatment when a dose-intensive immunochemotherapy was applied. Our findings suggest that patients with advanced DEL may not benefit from dose-intensified therapies, and therefore need highly discrete strategies. Disclosures Miura: Astellas Pharma Inc.: Honoraria; Celgene K.K.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; CHUGAI PHARMACEUTICAL CO. LTD: Honoraria; Kyowa Hakko Kirin CO., Ltd, Japan: Honoraria; Meiji Seika Pharma: Honoraria; Janssen Pharmaceutical K.K.: Honoraria. Hatta:Kyowa Hakko Kirin CO., Ltd, Japan: Honoraria; CHUGAI PHARMACEUTICAL CO. LTD: Honoraria; Celgene K.K.: Honoraria. Iriyama:Brystol-Myers K.K.: Honoraria. Takei:Kyowa Hakko Kirin CO., Ltd, Japan: Research Funding; Bristol-Myers K.K.: Research Funding; Nippon Kayaku Co.: Research Funding; Shionogi & Co.: Research Funding; Meiji Seika Pharma: Research Funding; Astellas Pharma Inc.: Research Funding; Janssen Pharmaceutical K.K.: Research Funding; TEIJIN PHARMA LIMITED: Research Funding; CSL Behring K.K: Research Funding; Japan Blood Products Organization: Research Funding; Sumitomo Dainippon Pharma Co.: Research Funding; TORII, PHAMACEUTICAL CO: Research Funding; Alexion Pharmaceuticals: Research Funding; YAKULT HONSHA CO., Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; TAIHO PHARMACEUTICAL CO., Ltd.: Research Funding; CHUGAI PHARMACEUTICAL CO. LTD: Research Funding.
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Bassi, Giulio, Luciano Pacelli, Cedric Mènard, Francesco Bifari, Luc Sensebé, Frederic Deschaseaux, Fabien Guilloton, et al. "Microporous Biphasic Calcium Phosphate Granules (MBCP®) Retain Immunological Properties of Bone Marrow-Derived Mesenchymal Stromal Cells and Promote Osteoblastic Differentiation." Blood 118, no. 21 (November 18, 2011): 1924. http://dx.doi.org/10.1182/blood.v118.21.1924.1924.
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Abstract Abstract 1924 Bone is among the most frequently transplanted tissue with about 1 million procedures annually in Europe. Allografts and autografts account for more than 80% of total graft volume, despite their considerable disadvantages, including the risk of disease transfer and immunologic rejection, limited supply of bone, costs and complications. Significant growth opportunities exist for synthetic bone grafts in association with mesenchymal stromal cells (MSC) from autologous or allogeneic sources as alternatives to biological bone grafts in orthopaedic and maxillofacial surgery. The objective of REBORNE is to perform clinical trials using advanced biomaterials and cells triggering bone healing in patients. To reach this goal, five phase I clinical studies with 20 patients have been planned in 12 clinical Centers spread in 8 European countries. Aim of the Immunological Unit of Reborne is to assess the MSC immunomodulatory properties in presence of the biomaterial used as scaffold for MSC delivery. All the functional experiments were performed in parallel, by comparing the effects of standard culture conditions and three-dimensional culture setting using MBCP (Biomatlante). Material and methods: Bone marrow MSC were provided from REBORNE Consortium Centers. To perform proliferation assays, different immune effector cells (T, B and NK cells) were stained with CFSE according to manufacturer's protocol. Active caspase-3 cell staining was used for survival quantification of immune effector cells after co-culture experiments. Differentiation potential was evaluated by culturing MSC with two different media containing either bone morphogenetic protein 4 (BMP4) or dexamethasone. After three weeks, osteogenic differentiation was quantified by qRT-PCR, alkaline phosphatase activity and alizarin red staining. Results: We found that primed MSC, pre-treated with the inflammatory cytokines IFNg and TNFa, displayed upregulation of HLA-ABC, CD54, CD106 and de novo expression of HLA-DR, both in standard culture conditions and in association with MBCP. Immune effector cells could be cultured and collected even in presence of MBCP and no significant differences were found between standard- (MSC + effector cells) and 3D-coculture conditions (MSC + MBCP + effector cells), in terms of immune effector cell proliferation. In both experimental conditions MSC suppressed T and NK cell proliferation (% suppression: MSC + T = 68.4; MSC + MBCP + T = 62.4; MSC + NK = 17.5; MSC + MBCP + NK = 20.2) and increased B cell proliferation (MSC + B = +13%; MSC + MBCP + B = +12.3%). In addition, immune effector cells viability was not affected by MBCP and MSC co-culture increased their survival even in presence of MBCP; in fact, in each culture condition the percentage of inhibition of T, B and NK cell apoptosis was higher than 20% in comparison to immune effector cells cultured without MSC. Dexamethasone and BMP4 were capable of inducing MSC differentiation into osteoblast-like cells, as confirmed by qRT-PCR analysis. We demonstrated that BMP4-based medium led to fully differentiated osteoblasts (Osterix+, RUNX2+, DLX5+ and alkaline-phosphatase+). Moreover, MBCP was more efficient in increasing osteoblastic differention as compared to standard culture conditions, as shown by the higher expression of Osteocalcin and Osterix. These data show that the association of MBCP and MSC does not affect MSC properties and suggest that it could be a treatment of choice of bone defects instead of allograft and autograft transplantation. Disclosures: No relevant conflicts of interest to declare.
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Lee, S. H., H. J. Oh, M. J. Kim, G. A. Kim, E. M. N. Setyawan, Y. B. Choi, S. Lee, J. X. Jin, A. Taweechaipaisankul, and B. C. Lee. "174 EFFECT OF HUMAN ENDOTHELIAL PROGENITOR CELLS ON IN VITRO MATURATION OF PORCINE OOCYTES AND PARTHENOGENETIC EMBRYO DEVELOPMENT COMPETENCE." Reproduction, Fertility and Development 29, no. 1 (2017): 195. http://dx.doi.org/10.1071/rdv29n1ab174.
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In oocyte maturation, hepatocyte growth factor and vascular endothelial growth factor (VEGF) contribute to promote granulosa cell proliferation and cumulus cell expansion. It is well known that human endothelial progenitor cells (hEPC), which are isolated from monocytes and macrophages, secrete a variety of growth factors, such as hepatocyte growth factor and VEGF, and improve the process of angiogenesis. Therefore, the aim of this study was to investigate the effects of hEPC on in vitro oocyte maturation and subsequent embryo development in pigs. To isolate and culture hEPC, human peripheral blood sample was collected from a healthy donor and peripheral blood mononuclear cells were separated. The peripheral blood mononuclear cells were seeded into flask with defined Keratinocyte-SFM-based medium and incubated at 37°C, 5% CO2. The hEPC were cultured and cryopreserved until use for co-culturing with porcine oocytes obtained from a local slaughterhouse ovaries. Cumulus-oocyte complexes were randomly cultured in 2 groups; 1) co-culturing with hEPC and 2) culturing without hEPC. Cumulus-oocyte complexes were cultured in the in vitro maturation (IVM) medium containing TCM-199 supplemented with 0.57 mM cysteine, 0.91 mM sodium pyruvate, 5 μL mL−1 of insulin-transferrin-selenium solution 100X (Invitrogen, Seoul, South Korea), 10% porcine follicular fluid, 10 IU mL−1 of eCG, and 10 IU mL−1 of hCG. After IVM, the first polar body extrusion was observed under the microscope. To evaluate embryo development competence, the matured oocytes were activated with electrical stimulus and cultured in porcine zygote medium-5 for 7 days. The cleavage and blastocyst formation rates were observed on Day 2 and 7, respectively. Also, blastocysts were stained with Hoechst 33342 and total blastocyst cell numbers were evaluated under a fluorescence microscope. As a result, the oocyte maturation rate or first polar body extrusion rate of the hEPC co-culture group (90.06 ± 0.75) was significantly higher than the control group (90.06 ± 0.75 v. 85.79 ± 0.59; P < 0.05). There was no significant difference between the hEPC co-cultured and the control groups in cleavage rate. However, a significant difference in blastocyst formation rate was observed between the hEPC co-cultured and the control groups (28.45 ± 4.92 v. 15.87 ± 2.27; P < 0.05), whereas total blastocyst cell numbers did not show significant difference between the 2 groups. The all data were analysed by unpaired t-test using GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). Values are means ± standard error of mean. In conclusion, the results in the present study demonstrated that co-culturing with hEPC improved the in vitro oocyte maturation and blastocyst formation rate. Also, we are underway in analysing the concentration of VEGF families in the hEPC co-culture medium after IVM. For further study, we will analyse the genes of the VEGF signaling pathway in the cumulus cells and matured oocytes derived from the 2 groups. This research was supported by Nature Cell (#550-20150030), global PH.D Fellowship Program through NRF funded by the Ministry of Education (NRF-20142A1021187), and Research Institute for Veterinary Science, the BK21 plus program.
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Dwina, Yayi, Ria Kodariah, and Endang S. R. Hardjolukito. "Identification of pathogenesis pathway in basal-like breast cancer based on mutant p53 protein and topoisomerase-IIα expression." Medical Journal of Indonesia 23, no. 4 (January 27, 2015): 197–202. http://dx.doi.org/10.13181/mji.v23i4.995.
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Background: Basal-like breast cancer is difficult to treat with standard regimen therapy, because it doesn’t express hormone receptors or epidermal growth factor receptors. Identification of oncogenesis pathway is expected to find molecules which can be used as target for therapy. One candidate molecule is topoisomerase-IIα whose expression is regulated by p53. This study aimed to compare the expression of mutant p53 proteins and topoisomerase IIα in basal-like and non basal-like breast cancer, and to determine the association between mutant p53 proteins and topoisomerase IIα in basal-like group.Methods: The samples were 40 formalin fixed paraffin embedded tissues from verified triple negative breast cancer tissue. The samples were divided into 2 groups, basal-like and non basal-like breast cancer, based on cytokeratin - 5 (CK-5) expression. Mutant p53 proteins and topoisomerase IIα were stained using immunohistochemistry method, scored and compared. Statistical test used SPSS software version 16 for descriptive statistics, kappa test, normality test, comparison of two mean, and categorical comparison.Results: Median (min-max) of mutant p53 protein expression in basal-like group was 21 (0-100), the non basal-like group was 2 (0-80), p = 0.061. Min-max of topoisomerase IIα in basal-like group was 263 (15-492), non basal-like group was 262 (0-481), p = 0.409. There was an association between mutant p53 positivity with breast cancer subtype (p = 0.027) and between mutant p53-topoisomerase IIα coexpression with breast cancer subtype (p = 0.018).Conclusion: Co-expression of mutant p53 with topoisomerase IIα has the role in one of the pathway of basal-like breast cancer pathogenesis.
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Demyda-Peyrás, S., M. Hidalgo, J. Dorado, and M. Moreno-Millan. "87 EVALUATION OF THE NUMERICAL CHROMOSOMAL ABNORMALITIES ON IN VITRO EARLY BOVINE EMBRYOS: EFFECT OF THE CELL CO-CULTURE WITH GRANULOSA CELLS." Reproduction, Fertility and Development 26, no. 1 (2014): 157. http://dx.doi.org/10.1071/rdv26n1ab87.
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Chromosomal numerical abnormalities (CNA) were described as a major cause of developmental failures in in vitro-produced (IVP) embryos. It has been described that CNA are influenced by the post-fertilization culture environment of the embryo. Furthermore, it was demonstrated that the use of different culture media affects the CNA rates. The addition of granulosa cells during early embryo development is a well-known procedure to simplify the culture of bovine IVP and cloned embryos. This technique avoids the use of culture environments saturated with N2 (tri-gas chambers). The aim of this study was to determine the effect of the addition of granulosa cells in the chromosomal abnormalities of IVP cattle embryos. Cumulus–oocyte complexes (COC) were matured in TCM-199 medium, supplemented with glutamine, sodium pyruvate, FSH, LH, oestradiol, and gentamicin during 20 h at 38.5°C in a 5% CO2 humid atmosphere. Subsequently, matured oocytes were fertilized in IVF-TALP medium using 1 × 106 spermatozoa mL–1, selected through a Percoll gradient centrifugation. After fertilization, zygotes were divided in 2 groups and cultured in TCM-199 medium for 48 h, with (TCM-GC) or without (TCM) the addition of 1 × 106 granulosa cells. These cells were obtained by centrifuging and washing the follicular fluid remaining from searching dishes and adjusted to the working concentration. After culture, a total of 106 early embryos (72 hpi) were cytogenetically evaluated following our standard laboratory techniques. Embryos showing normal development were individually fixed onto a slide, disaggregated into blastomeres with acetic acid, and stained with Giemsa solution. Chromosomal numerical abnormalities were evaluated by direct observation at 1250× magnification in a brightfield microscope. Percentage of normal diploid embryos (D) and abnormal haploid (H), polyploid (P), or aneuploid (A) embryos were determined. Results were statistically compared between treatments using a Z test for proportions. Results were: D = 81.4%, H = 7.2%, P = 7.2%. and A = 3.6% in TCM and D = 84.3%, H = 3.9%, P = 9.8%, and A = 1.9% in TCM-GC. No significant differences (P > 0.05) were found between culture media in the chromosomal abnormality rates. According to our results, the use of somatic cells in co-culture during embryo development did not influence the appearance of abnormal complements in the produced embryos. This would allow the use of GC as a potential complement to simplify the techniques used in the culture of bovine embryos until Day 3.
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Thaweboon, Sroisiri, Passiri Nisalak, Boonyanit Thaweboon, Pornrachanee Sawaengkit, Plang Ngern Saksit, and Rattiporn Kaypetch. "Antimicrobial Activity of Type III Dental Gypsum Incorporated with 3-iodo-2-propynyl-butylcarbamate and its Physical Properties." Advanced Materials Research 1052 (October 2014): 322–26. http://dx.doi.org/10.4028/www.scientific.net/amr.1052.322.
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Dental impressions have been considered to be potential sources of microbial contamination from patients’ blood and saliva to dental personnel and prostheses fabricated on gypsum casts. Thus, the development of dental gypsum with antimicrobial activity to reduce cross-contamination between patients and laboratory personnel is needed. This study aims to evaluate the influence of incorporation of 3-iodo-2-propynyl-butylcarbamate (IPBC) into type III dental gypsum on its antimicrobial activities and physical properties such as dry compressive strength and setting time. Type III dental gypsum (The Siam Moulding Plaster Co., Ltd, Thailand) incorporated with 3 concentrations of IPBC (0.01%, 0.005% and 0.001% w/w) was tested compared with the control, gypsum without disinfectant. Microorganisms tested were Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 27853 and Candida albicans ATCC 1023. One hundred μL of each microbial suspension (108 CFU/mL) was dropped on hydrocolloid impression (Jeltrate; Caulk/Dentsply, Milford, DE) and left dry. All types of gypsum mixes were prepared and poured into the impressions and allowed to set for 60 min. Then the gypsum samples were removed and the microbial contact surfaces were imprinted on Brain Heart Infusion agar plates. After incubation, colonies appeared on agar were gram-stained and counted. The dry compressive strength and setting time were tested in accordance with International Standard (ISO) 6873: 1998 (E). Dental gypsum containing IPBC showed antimicrobial activity against all tested microorganisms with the percentage of microbial reduction ranging from 19.4% to 70.6%. Among all types of dental gypsum, no significant differences in dry compressive strength and setting time were observed. The newly developed type III dental gypsum incorporated with IPBC had antimicrobial effects against all tested microorganisms. The physical properties of the modified dental gypsum were within the ISO standards. However, further investigation on other properties such as dimensional stability, detail reproduction and clinical usage are still needed.
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Gozhenko, A., O. Bestanchuk, O. Kaschenko, and T. Narbutova. "Cumulative cardiotoxic effect of bleomycin in experiment." Journal of Education, Health and Sport 11, no. 6 (June 30, 2021): 301–8. http://dx.doi.org/10.12775/jehs.2021.11.06.033.
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The aim of the study was to evaluate the cumulative toxic effect of bleomycin under experimental conditions
Material and Methods. The study was conducted in the Research Institute of Transport Medicine during 2016-2021. The experimental model of the cardiotoxic effect of the bleomycin was performed using the medication "Bleocin" manufactured by Nippon Kayaku Co., Ltd. (Japan). According to the task, the study was performed on 10 mature rats of both sexes of the Wistar line with a body weight of 237 ± 20 g.
Rats were housed in standard vivarium conditions of Odessa National Medical University. Animals were divided into 2 groups: experimental group (n = 5) and control (n = 5). Bleomycin animals of the experimental group were obtained intraperitoneally at a dose of 0.5 IU / kg on 1st and 8th days. Withdrawal of animals from the experiment was performed on the 5th, 14th and 28th day of the experiment, followed by morphological and morphometric examination.
The material after weighing and morphometry was fixed with a neutral 10% formalin solution and poured into paraffin. Histological sections were stained with hematoxylin-eosin, MSB, Van Gizon. Performed light microscopy.
After two weeks there was a decrease in myocardial weight by 7-10% from baseline, there were pronounced dystrophic changes in the myocardium. Repeated administration of bleomycin has a cumulative cardiotoxic effect leading to irreversible changes in the myocardium and endothelial dysfunction clinically manifested by heart attack, vascular pathology at significant cumulative doses of bleomycin. Morphofunctional changes of the right ventricle are prominent and considered to be pathognomic for bleomycin toxicity.
Conclusions: 1. Bleomycin has a cumulative toxic effect on the myocardium of mammals
2. The severity of the cardiotoxic effect of bleomycin is proportional to the tissue concentration of the drug, which is proportional to the duration of exposure
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Kurmi, Roshan, Ramanuj Rauniyar, Krishna Das Manandhar, and Birendra Prasad Gupta. "Evaluation of the XpertMTB/RIF for the Diagnosis of Pulmonary Tuberculosis Among the Patients Attending DOTS Center Parsa District of Nepal." Nepal Journal of Biotechnology 4, no. 1 (December 31, 2016): 26–32. http://dx.doi.org/10.3126/njb.v4i1.16343.
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Tuberculosis diagnosis and monitoring rely in Sputum microscopy of National Tuberculosis Programme, Nepal because of its low cost and easier to perform. Direct sputum microscopy is popular worldwide. Currently, there are 533 microscopy centres catering for sputum microscopy examination throughout the country. Most of the microscopy centres are established within government jurisdiction and remaining are established as non-governmental organization as well as private sectors.A cross-sectional study was conducted from July 2013 to January 2015. A total of 2091 patients were enrolled in the study who were attending the DOTS Centre in Parsa District of Public Health Office, Nepal. Smears stained with ZN stain methods were examined microscopically followed by the GeneXpert MTB/RIF assay.Out of 2091 suspected pulmonary TB patients enrolled for sputum microscopy and GeneXpert MTB/RIF for the confirmation of TB, the 1301(62.21%) were male and 790 (37.78%) were female. The maximum TB cases were from Parsa district (555, 26.5%). The comparative study of different diagnostic tools reveals the sensitivity of MTB/RIF was 95.50% (91.87, 97.82) and significantly higher than smear microscopy performed on the same fluid, which had a sensitivity of 61.97% (55.41, 68.21). Five of 127 smear-negative cases had MTB/RIF-positive un-centrifuged sputum, resulting in a specificity of 81.23% (75.95, 85.78), which was similar to smear microscopy 98.29 % (97.34, 98.97; p=0.121). The positive predictive value (PPV) and negative predictive value (NPV) of MTB/RIF were 96.85% (93.61, 98.72) and 94.95 % (93.52, 96.14), respectively. HIV co-infection did not impact sensitivity, specificity or liquid culture time to positivity (TTP). When MTB/RIF accuracy was evaluated using composite reference standard culture positivity from sputum, the sensitivity and specificity were similar to those obtained in the primary analysis using either definite TB versus possible and non-TB combined; definite and possible TB combined versus non-TB.
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Lim, Ki-Byung, Jannie Wennekes, J. Hans de Jong, Evert Jacobsen, and Jaap M. van Tuyl. "Karyotype analysis of Lilium longiflorum and Lilium rubellum by chromosome banding and fluorescence in situ hybridisation." Genome 44, no. 5 (October 1, 2001): 911–18. http://dx.doi.org/10.1139/g01-066.
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Detailed karyotypes of Lilium longiflorum and L. rubellum were constructed on the basis of chromosome arm lengths, C-banding, AgNO3 staining, and PI-DAPI banding, together with fluorescence in situ hybridisation (FISH) with the 5S and 45S rDNA sequences as probes. The C-banding patterns that were obtained with the standard BSG technique revealed only few minor bands on heterologous positions of the L. longiflorum and L. rubellum chromosomes. FISH of the 5S and 45S rDNA probes on L. longiflorum metaphase complements showed overlapping signals at proximal positions of the short arms of chromosomes 4 and 7, a single 5S rDNA signal on the secondary constriction of chromosome 3, and one 45S rDNA signal adjacent to the 5S rDNA signal on the subdistal part of the long arm of chromosome 3. In L. rubellum, we observed co-localisation of the 5S and 45S rDNA sequences on the short arm of chromosomes 2 and 4 and on the long arms of chromosomes 2 and 3, and two adjacent bands on chromosome 12. Silver staining (Ag-NOR) of the nucleoli and NORs in L. longiflorum and L. rubellum yielded a highly variable number of signals in interphase nuclei and only a few faint silver deposits on the NORs of mitotic metaphase chromosomes. In preparations stained with PI and DAPI, we observed both red- and blue-fluorescing bands at different positions on the L. longiflorum and L. rubellum chromosomes. The red-fluorescing or so-called reverse PI-DAPI bands always coincided with rDNA sites, whereas the blue-fluorescing DAPI bands corresponded to C-bands. Based on these techniques, we could identify most of chromosomes of the L. longiflorum and L. rubellum karyotypes.Key words: fluorescence in situ hybridisation, FISH, 5S rDNA, 45S rDNA, C-banding, reverse PI-DAPI banding.
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Sharma, Shilpa, Renata Pereira, Elizabeta Nemeth, Mark Hanudel, Joachim Ix, Isidro Salusky, and Tomas Ganz. "Serum Ferritin Reflects Bone Marrow Iron Stores in Children with End Stage Kidney Disease." Blood 142, Supplement 1 (November 28, 2023): 1090. http://dx.doi.org/10.1182/blood-2023-182483.
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Background: Iron deficiency is common in children with end-stage kidney disease (ESKD) but diagnosing iron deficiency in ESKD is challenging as serum indicators of iron stores may be influenced by inflammation and other factors. We examined which of the clinically used serum iron indices were predictive of bone marrow iron stores (the gold standard) in dialysis-dependent children and young adults with ESKD, in the setting of increased nutritional requirements and co-existing inflammation. Methods: This cross-sectional study enrolled 71 clinically-stable children and young adults (age range 2 to 28 years) receiving dialysis, who underwent bone biopsy for CKD-mineral bone disorders between 2007 through 2011. Congenital anomalies of the kidney and urinary tract (CAKUT) and hereditary diseases (63.3%) were the main causes of renal failure, followed by primary glomerulonephritis (36.7%). Bone samples were stained with Perls' Prussian blue and independently interpreted by a pathologist unaware of the patients' iron parameters and clinical status. Staining was scored absent vs present for receiver operator curve (ROC) analysis. In ROC analysis, the ability of serum ferritin to predict bone sample iron stores was compared with serum hepcidin, transferrin saturation (TSAT), and clinical guideline-based iron deficiency cut-offs for serum iron, TSAT, and their combination. We generated optimal cut off points using the Youden's J statistic. Results: Mean age was 17.2 ± 4.4 years, and 30% of subjects were female. Median dialysis vintage was 1.2 (IQR 0.7, 2.0) years, and 56% were supported by peritoneal dialysis. Mean hemoglobin was 12.4 ± 1.7 g/dl, and 35% were receiving iron supplementation at the time of biopsy. Based on gold standard of depleted bone iron stores, 46.5% patients were iron-deficient. As a predictor of bone marrow iron staining, serum ferritin provided the highest area under the ROC curve compared to other tests (Table), but at the usual clinical ferritin cutoff (100 ng/ml) only 22% of true iron-deficient subjects were diagnosed compared to 52% of true iron-deficient subjects using a higher optimal cutoff point for ferritin (188 ng/ml). Conclusions: In children with ESKD, iron deficiency as determined by the gold standard bone iron staining was highly prevalent (close to 50%), and serum ferritin provided the best prediction of bone sample iron stores. Optimal ferritin cutoff for decision-making about iron administration should reflect the current relative risks and benefits of iron therapy. Future trials testing higher ferritin cut-offs and patient centered outcomes in children with ESKD are timely.
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Gibson, Amber, Pariya Sukhumalchandra, Celine Kerros, Na Qiao, Anne Philips, Mao Zhang, Sami Alkoutami, et al. "Abstract 2995: Cross-presented serine proteases as immunotherapy targets for lung cancer and osteosarcoma." Cancer Research 83, no. 7_Supplement (April 4, 2023): 2995. http://dx.doi.org/10.1158/1538-7445.am2023-2995.
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Abstract Background: Immunotherapy is the standard of care for many solid tumor malignancies. However, due to a paucity of known tumor antigens in non-small cell lung cancer (NSCLC) and osteosarcoma (OS), identification of novel immunogenic antigens is needed. Our group has experience with neutrophil serine proteases (NSP) derived from the azurophilic granules of polymorphonuclear leukocytes (PMN), specifically, cathepsin (CG), proteinase 3 (P3), and neutrophil elastase (NE). We have identified two HLA-A2 (HLA-A*0201)-restricted nonameric peptides: CG1 (FLLPTGAEA), derived from CG, and PR1 (VLQELNVTV), derived from NE and P3. We have previously shown that CG1 and PR1 are effective targets in hematological malignancies and that PR1 can be a target for some solid tumor malignancies. Here, we extend our findings to demonstrate that CG1 and PR1 can be immunotherapeutic targets for NSCLC and OS. Methods: Healthy donor PMN were isolated from buffy coats using double Ficoll gradient centrifugation. To evaluate uptake, malignant cells were pulsed with PMN lysates, harvested, permeabilized, and stained with anti-CG, anti-NE or anti-P3. To evaluate cross-presentation, cells were cultured with PMN lysates, harvested and stained with CG1 TCR-like antibody, or PR1 TCR-like antibody (8F4). Flow cytometry was performed using the Fortessa X-20 Cell Analyzer. CG1- or PR1-specific cytotoxic T-lymphocyte (CTL) were expanded from healthy donor peripheral blood mononuclear cells. Standard cytotoxicity assays were used to evaluate the lysis of target cells co-cultured with CG1- or PR1-specific CTL. Results: We have previously demonstrated that NE and P3 are taken up and cross-presented by NSCLC and become targets for PR1-CTL, thus in this study, we investigate if NSCLC cells take up CG, and whether OS cells take up NE and P3, resulting in enhanced cytotoxicity by peptide-specific CTLs. We first employed The Cancer Cell Line Encyclopedia to show the absence of NSP’s in NSCLC and OS cell lines. RT-PCR confirmed lack of endogenous CG mRNA expression in NSCLC and OS, and lack of NE and P3 in OS cell lines. Next, we analyzed antigen cross-presentation and showed that NSCLC cells take up and cross-present CG, leading a 13.3-fold increase in CG1-CTL killing of NSCLC cells pulsed with CG versus non-pulsed cells. In addition, OS cells take up and cross-present NE and P3, with a 13.1-fold increase in PR1-CTL killing of OS cells that were pulsed with NE and P3 vs. non-pulsed cells. Conclusion: Our data define two novel HLA-A2 restricted peptides that can be targeted in NSCLC and OS. Specifically, CG1 and PR1 are presented on the surfaces of NSCLC and OS, increasing their susceptibility to killing by CG1 and PR1 specific CTL. Our work represents a novel link between innate and adaptive immune responses in solid tumors and identifies potential antigen targets for CTL, peptide vaccines, and TCR-like antibodies in the setting of NSCLC and OS. Citation Format: Amber Gibson, Pariya Sukhumalchandra, Celine Kerros, Na Qiao, Anne Philips, Mao Zhang, Sami Alkoutami, Scott Semelsberger, Yoshimi Lu, Lauren Byers, Ze Tian, Chunhua Shi, Jun Yan, Dongxing Zha, Alejandro Marinos, Anne Sergeeva, Hong He, Lisa St. John, Jeffrey J. Molldrem, Gheath Alatrash. Cross-presented serine proteases as immunotherapy targets for lung cancer and osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2995.
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Sewerin, P., L. Dötsch, D. Truhm, D. Abrar, and S. Nebelung. "THU0062 FUNCTIONAL MR IMAGING OF HUMAN MENISCUS IS ASSOCIATED WITH HISTOLOGICAL DEGENERATION." Annals of the Rheumatic Diseases 79, Suppl 1 (June 2020): 243. http://dx.doi.org/10.1136/annrheumdis-2020-eular.6403.
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Background:In OA, there is a close association of meniscus and cartilage pathologies. Meniscus degeneration and lesioning are critical risk factors for development of early OA. Hence, thisex-vivostudy assessed the responses to standardized loading of human meniscus samples as a function of degeneration and based on changes in their T1, T2 and T1ρ maps (as surrogate parameters of the tissue’s functionality).Objectives:Can meniscus functionality be visualized by serial quantitative MRI mapping technics?Methods:During total knee replacements, 45 meniscus samples of variable degeneration were harvested from the center of the lateral meniscus body (Fig. 1a1-a3). After preparation to standard, samples were subject to force-controlled loading using an MRI-compatible lever device that created compressive loading by torque ((Fig. 1a4-a5). For each sample and loading position, MRI measurements (as detailed below) were performed in the unloaded (δ0) and loaded configurations, i.e. loaded to 2 bar (δ1, 37.1 N compressive force, 0.67 Nm torque) and to 4 bar (δ2, 69.1 N, 1.24 Nm). Throughout all loading positions, morphological and quantitative imaging was performed using Proton Density-weighted and T1, T1ρ, and T2 mapping sequences (3.0 T, Achieva, Philips) based on standard turbospin-echo, inversion-recovery, spin-lock multi-gradient-echo, and multi-spin-echo sequences. For reference purposes, histological (i.e. Pauli classification) and biomechanical measures (i.e. Elastic Modulus) were obtained for each sample. Based on Pauli sum scores, samples were trichotomized as grossly intact, (n=14), mildly degenerated (n=16), and moderate-to-severely degenerated (n=15).Figure 1.Preparation of meniscus samples and details of the MRI-compatible loading device. The lateral meniscus (a1) was cut to standard size by use of a dedicated cutting block (a2) to eventually obtain lateral meniscus samples (from the body region) of standard dimensions (a3). These samples were then placed in a dedicated MRI-compatible loading device for pressure-controlled, quasi-static and torque-induced loading under simultaneous MR imaging (a4). Two parallel support beams allowed standardized positioning in the MRI scanner‘s bore (a5).Results:Morphologically, loading induced deformation and flattening in all samples (Fig. 2a). For T1, homogeneous loading-induced decreases in all samples were found, irrespective of degeneration (Fig. 2b). For T1ρ, increases in the apical zones of intact samples were observed, and decreases in degenerated samples (Fig. 2c). For T2, changes were ambiguous and incoherent (Fig. 2d).Figure 2.Serial morphological images and functional maps of histologically moderately degenerative human meniscus as a function of force-controlled loading. Serial PDw (a), T1 (b), T1ρ (c), and T2 maps (d) are displayed at increasing loading intensity (δ0: unloaded [a1-d1]; δ1: loaded to 2 bar [a2-d2]; δ2: loaded to 4 bar [a3-d3]). Histologically, this sample demonstrated signs of severe surface desintegration and disruption. Pauli sum score 12, i.e. moderate to severe degeneration (Pauli Grade III). In b – d, color-coded parameter value maps are overlaid onto the corresponding morphological images. Histological sections are stained with Hematoxylin-Eosin (e1) and Safranin O (e2).Conclusion:Meniscus functionality may be visualized using serial quantitative MRI mapping techniques. T1ρ may provide an imaging biomarker of relevant intra-tissue adaptations that seem to be associated with histological degeneration. The perspective evaluation of meniscus functionality may be indicative of incipient or manifest load transmission failure to the adjacent cartilage layer.Disclosure of Interests:Philipp Sewerin Grant/research support from: AbbVie Deutschland GmbH & Co. KGBristol-Myers Squibb Celgene GmbHLilly Deutschland GmbHNovartis Pharma GmbH Pfizer Deutschland GmbHRheumazentrum Rhein-Ruhr, Consultant of: AMGEN GmbH AbbVie Deutschland GmbH & Co. KG Biogen GmbHBristol-Myers Squibb Celgene GmbH Chugai Pharma arketing Ltd. / Chugai Europe GmbHHexal Pharma Janssen-CilagGmbH Johnson & Johnson Deutschland GmbHLilly Deutschland GmbH / Lilly Europe / Lilly Global Novartis Pharma GmbH Pfizer Deutschland GmbH Roche Pharma Rheumazentrum Rhein-Ruhr Sanofi-Genzyme Deutschland GmbH Swedish Orphan Biovitrum GmbH UCB Pharma GmbH, Speakers bureau: AMGEN GmbH AbbVie Deutschland GmbH & Co. KG Biogen GmbHBristol-Myers Squibb Celgene GmbH Chugai Pharma arketing Ltd. / Chugai Europe GmbHHexal Pharma Janssen-CilagGmbH Johnson & Johnson Deutschland GmbHLilly Deutschland GmbH / Lilly Europe / Lilly Global Novartis Pharma GmbH Pfizer Deutschland GmbH Roche Pharma Rheumazentrum Rhein-Ruhr Sanofi-Genzyme Deutschland GmbH Swedish Orphan Biovitrum GmbH UCB Pharma GmbH, Lisa Dötsch: None declared, Daniel Truhm: None declared, Daniel Abrar: None declared, Sven Nebelung: None declared
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Zakharov, Oleg D., Ekaterina U. Rybalkina, Alla A. Stavrovskaya, and Maya Volkova. "Prognostic Significance of Multidrug Resistance Proteins Expression in Acute Myeloid Leukemia." Blood 108, no. 11 (November 16, 2006): 4360. http://dx.doi.org/10.1182/blood.v108.11.4360.4360.
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Abstract Background: Conventional induction chemotherapy induces complete remission (CR) in 65–75% of adults with de novo acute myeloid leukemia (AML), and 15–20% of the patients have refractory disease. We investigated the prognostic significance of multidrug resistance proteins (P-glycoprotein (Pgp), BCRP, MRP1 and LRP) expression on AML blast cells before treatment. Methods: We included in analysis 30 patients (pts) with de novo AML. Expression of multidrug resistance proteins (MDR) was detected by indirect immunofluorescence technique and flow cytometry on bone marrow blast cells before chemotherapy. Expression of MDR proteins was considered as positive if at least 25% of the blast cells were stained by anti-MDR protein antibody. All pts received standard induction therapy (cytarabine, etoposide and idarubicin or daunorubicine). Results: Blast cells was defined as Pgp-positive in 64.3% of cases, BCRP+ in 42.9%, MRP1+ in 46.4%, and LRP+ in 64.3% of cases. After induction therapy 20 (66,7%) pts achieved CR and 10 pts (33.3%) were resistant. MDR proteins expression was observed more frequently in resistant group, then in a sensitive one (70% vs 61% for Pgp, 70% vs 33% for MRP1, 100% vs 44% for LRP, 80% vs 22% for BCRP, respectively), difference for LRP and BCRP was statistically significant (p=0.004). Blast cells of all resistant pts expressed 2–4 MDR proteins (all studied proteins − 40%, 3 of them − 40% and 2 proteins − 20%). In a group of pts archived CR the blast cells expressed 3 proteins only in 2 cases (10%), and the expression of all 4 proteins we observed only in 1 patient (5%) with very short CR duration (3 months). Other pts from this group express only one studied protein. According to chromosome analysis 18.2% of pts had favorable, 50% - intermediate and 31.8% unfavorable cytogenetic. Blast cells of all pts in cytogenetically unfavorable group expressed more then 1 protein, 3 or 4 MDR proteins expressed in 71,4% of cases. In favorable and intermediate cytogenetic groups blast cells expressed 1or 2 MDR proteins in 73,2% of cases, 3 or 4 proteins in 20%. Conclusions: The expression of MDR proteins in AML has a prognostic value with respect to CR achievement in pts receiving standard antracycline-Ara-C regimens. The detection of any single protein didn’t have prognostic significance, only co-expression of 2 and more proteins predict unfavorable treatment outcome. We observed a correlation between the cytogenetic and the MDR phenotype.
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Odeh, Mary, Alice Amachree, Adetutu Olubunmi Obulor, and Eme Efioanwan Orlu. "Critical Evaluation of the Role of Lycopene on Reproduction of Male Wistar Rats Exposed to Dimethoate." Asian Journal of Biology 18, no. 2 (May 4, 2023): 1–10. http://dx.doi.org/10.9734/ajob/2023/v18i2337.
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Aim: This study was aimed at evaluating the role of Lycopene on reproduction of male Wistar rats exposed to Dimethoate.
Experimental Design: A completely randomized experimental design using standard methods for analysis.
Location and Duration of Study: This study was carried out in the Animal house, Department of Animal and Environmental Biology of Rivers State University, Nkpolu-Oroworukwo Port Harcourt, Nigeria. GPS 4o47'50''N 6o58'49''E. This study lasted for 21days.
Methodology: Thirty male rats were randomly selected into five (5) groups A-E (6 animals per group). Groups B, C and D were gavaged 10, 20, 30 mg/kg/bw/day of dimethoate respectively, and co-administered Lycopene at 10mg/animal daily. Group E were administered 30mg/kg/bw/day of dimethoate without Lycopene. All animals were allowed access to cool clean water and standard rat pellet ad libitum. Twenty-four hours to the termination of the experiment, feed was withdrawn from the animals. Blood samples were collected by ocular puncture into heparinized tubes for hormonal analysis. Hormones such as Testosterone, Progesterone, estradiol, Follicle Stimulating Hormone (FSH) and luteinizing hormone (LH) were analysed based on the manufacturer’s instruction using Randox Monza assay kit. For Histopathological analysis of the testis, 0.5g of the testis was fixed in 10% formalin and sectioned with a digital Microtome (AO spencer No. 820) at 5μm thick. Histological sections were mounted on clean glass slides and stained with Hematoxylin and Eosin (H&E). The slides were viewed at X400 magnification (modified Orlu, 2014). Statistical analysis was carried out using one-way Analysis of Variance (ANOVA) and expressed as their respective units. Where significant differences were found, Pair-wise comparisons conducted with Tukey test using SPSS 22 software.
Results: It was observed that the level of all hormone considered decreased significantly with an increase in Dimethoate exposure despite the administration of lycopene. However, administration of dimethoate only resulted in a significant (p<0.05) reduction in the concentration of all hormones compared with groups given lycopene. The massive cellular degeneration observed in the seminiferous epithelium of experimental animals exposed to higher concentrations (20 and 30mg/kg/bw/day) of Dimethoate is indicative of a targeted action of the pesticide on male reproductive organ. The antioxidant property of Lycopene is clearly visible in testicular cross section of animals in groups given lycopene as gradual regeneration of both mitotic and meiotic spermatogenic elements were evidence.
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Lucas-Hahn, A., B. Petersen, M. Nowak-Imialek, U. Baulain, R. Becker, H. M. Eylers, K. G. Hadeler, P. Hassel, and H. Niemann. "122 A New Maturation Medium Improves Porcine Embryo Production In Vitro." Reproduction, Fertility and Development 30, no. 1 (2018): 200. http://dx.doi.org/10.1071/rdv30n1ab122.
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Recently (Spate et al. 2017 Reprod. Fertil. Dev. 29, 150), a new medium [TCM-199 supplemented with hCG 10 IU, pregnant mare serum gonadotropin (PMSG) 10 IU mL−1, fibroblast growth factor (FGF) 40 ng mL−1, leukemia inhibitory factor (LIF) 2000 U mL−1, IGF-1 20 ng mL−1, epidermal growth factor (EGF) 10 ng mL−1], termed FLI medium, was demonstrated to improve porcine oocyte maturation in vitro. The effects on embryo development and quality have not yet been investigated. The purpose of the present study was to compare the FLI medium in porcine in vitro embryo production (IVP) with our standard maturation medium (DMEM supplemented with 10 IU mL−1 PMSG and hCG, 50 ng mL−1 EGF, 100 ng mL−1 IGF1, and 5 ng mL−1 FGF). Briefly, gilt oocytes were collected via aspiration of follicles from abattoir ovaries and matured for 44 h in either FLI or standard DMEM medium at 39°C, 5% CO2 in humidified air. In vitro fertilization was performed with freshly ejaculated sperm (250,000 mL−1) of a multi-transgenic boar (GGTA1-KO/hCD46/hCD55/hCD59/hHO-1/hA20) by co-incubation with the matured oocytes in PGMTac4 medium for 4 h. Zygotes were washed twice and then cultured for 6 days in PZM3 medium. Development to the blastocyst stage was recorded at Day 6 of culture. Blastocysts were fixed and Hoechst33342 stained for counting the nuclei. Each of the experiments was repeated 3 times. In a second step, Day 5 blastocysts derived from the FLI medium were transferred to synchronized pubertal gilts to test the in vivo developmental competence of the IVF embryos. Maturation of oocytes in FLI medium resulted in a significantly higher blastocyst rate (49.3 vs. 13.5; P ≤ 0.001, Chi-squared test) and nuclei number (41.3 ± 12.2 vs. 35.3 ± 10.8; P ≤ 0.001, one-way ANOVA) compared with the standard medium, whereas the cleavage rate was not affected. Transfer of Day 5 blastocysts (average 35 embryos/recipient) derived from the FLI system using 8 recipients resulted in 7 pregnancies (87.5%) as determined by ultrasound scanning on Day 25 of gestation. At the time of writing, one recipient had delivered 5 healthy piglets after a gestation length of 114 days. Results indicate that the FLI medium significantly improves blastocyst rates and the cell number of the resulting blastocysts (Table 1) and yields pig IVF embryos with a high developmental capacity in vivo. By producing high-quality porcine embryos, this FLI-based IVF system provides an efficient method to modify the porcine genome by cytoplasmic microinjection of CRISPR/Cas molecules into IVF-derived zygotes. Table 1.Results of maturation of oocytes in FLI medium compared with DMEM
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Korban, Schuyler S., Wannasiri Wannarat, Charlotte M. Rayburn, Tatiana C. Tatum, and A. Lane Rayburn. "Genome size and nucleotypic variation in Malus germplasm." Genome 52, no. 2 (February 2009): 148–55. http://dx.doi.org/10.1139/g08-109.
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The genus Malus has anywhere between 25 and 33 species along with several subspecies. Malus species as well as clones within the same species have varying ploidy levels, as these are more than likely collected from different trees and (or) from different locations. In recent years, large numbers of Malus germplasm accessions have been collected and maintained at the United States National Germplasm Clonal Repository; however, genome sizes of this material have not yet been determined. In this study, leaf tissues from young grafted trees of 100 Malus species and hybrids growing in a nursery at the University of Illinois were collected and immediately used for extracting nuclei. Leaf tissues from apple and maize line W-22, used as an internal standard, were co-chopped and prepared for flow cytometric analysis. Apple nuclei were stained with propidium iodide, an intercalating dye, and a minimum of 8000 nuclei per sample were analyzed. Mean fluorescence of apple nuclei was then determined. A total of four replications per sample was used. Among 100 Malus accessions analyzed, one tetraploid, three triploid, and 96 diploid genotypes were identified. Significant differences in genome size were identified among the three ploidy types observed and also within diploid genotypes. The 2C mean value for tetraploids was 3.13 pg and ranged from 2.27 to 2.41 pg for triploids, whereas 2C values for diploids ranged between 1.44 and 1.72 pg. In addition, leaf impressions of young, fully expanded leaves were collected from young trees of 10 selected genotypes based on their ploidy and flow cytometric analysis and used to measure the nucleotypic parameter stomatal length. Ten stomata were measured per slide, three slides were analyzed per leaf, and three leaves were analyzed per accession. Overall, mean length of stomata ranged between 19.47 μm (diploid) and 27.6 μm (tetraploid), indicating that stomatal length in a tetraploid Malus genotype was 1.4-fold higher than that of a diploid genotype. A positive correlation between genome size and the nucleotypic parameter stomatal length was observed.
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Chen, Haiming, Mingjie Li, Suzie Vardanyan, Jillian Gottlieb, Cathy Wang, Kevin Delijani, Joseph Ben-Zvi, et al. "Increased M2 Macrophages and Higher Levels Of The Tribble Family Member Trib1 In Monocytes Are Associated With Progressive Disease In Multiple Myeloma (MM) Patients." Blood 122, no. 21 (November 15, 2013): 3127. http://dx.doi.org/10.1182/blood.v122.21.3127.3127.
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Abstract Macrophages consist of two subgroups, M1 and M2. M1 macrophages are pro-inflammatory cells against bacterial and viral infections whereas M2 macrophages are anti-inflammatory and associated with tumor progression. The mammalian tribble (Trib) family of genes, Trib1, Trib2 and Trib3, encode pseudokinase proteins that have important roles in monocyte/macrophage proliferation, differentiation, and apoptosis. Trib1 is a critical factor that induces M2 macrophage differentiation in the bone marrow (BM). First, we investigated the proportion of M1 and M2 macrophages in BM mononuclear cells (MCs) from multiple myeloma (MM) patients with progressive disease or in remission using flow cytometric analysis. The percentage of M2 (CD36+/CD86-) macrophages in BM was significantly increased in MM patients with progressive disease (n=15) compared to those in remission (n=5; P<0.05) whereas there was no difference in the percentage of M1 (CD86+/CD14+) macrophages in BM derived from MM patients with progressive disease compared to those in remission. Using immunohistochemical (IHC) analysis, the proportion of M2 macrophages was also determined in MM BM biopsies from patients with progressive disease and remission. The samples were cut into five-micrometer sections and double stained with two antibodies following a standard IHC protocol. IHC demonstrated that the percentage of M2 macrophages (CD36+/CD14+) was markedly increased in BM sections from MM patients with progressive disease compared to those in remission. In contrast, the percentage of M1 (CD86+/CD14+) macrophages was not different among those patients with progressive disease compared to those in remission. Next, we analyzed Trib1, Trib2 and Trib3 gene expression in BMMCs obtained from MM patients with progressive disease or in remission. RT-PCR results showed Trib1 expression levels were much higher among patients with progressive disease compared to those in remission. In contrast, the expression Trib2 and Trib3 was not related to the MM patient's clinical status. To determine whether MM tumor cells affected monocyte/macrophage differentiation and Trib gene expression, we co-cultured fresh MM tumor cells with purified healthy human monocytes. BMMCs from MM patients were co-cultured with human monocytes from normal subjects using Transwell plates and the percentage of M1 and M2 macrophages was determined using flow cytometric analysis following 2, 5 and 7 days of culture. The percentage of M2 cells increased whereas the proportion of M1 cells decreased. Gene expression of Trib1, Trib2 and Trib3 was analyzed using RT-PCR following 2, 5, and 7 days of co-culture. The expression of Trib 1 increased during the 7 days of co-culture whereas the expression of Trib2 and Trib3 did not change. Moreover, when direct cell-to-cell contact occurred between the MM cells and the monocytes, the percentage of M2 macrophages (CD36+) markedly increased after 7 days of incubation. We have shown that MM cells induce monocytes to increase Trib1 gene expression, which stimulates M2 differentiation in monocytes. M2 cells, in turn, induce tumor progression, providing a positive feedback loop on Trib1 expression, monocyte differentiation and tumor cell growth. Overall, we propose that Trib1 may be considered as a potential novel therapeutic target for the treatment of MM. Disclosures: No relevant conflicts of interest to declare.
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Bettelli, Alice, Rita Ruggiano, Silvia Bocchi, Laura Rocchi, Andrea Faenza, Enrica Borsi, Carolina Terragna, et al. "Inter-Cell Networking Profiling Enables Comprehensive Characterization of Immune-Mediated Activity of Anti-CD38 Therapy through Ex-Vivo Analysis of Multiple Myeloma Patients." Blood 134, Supplement_1 (November 13, 2019): 3372. http://dx.doi.org/10.1182/blood-2019-126894.
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There has been a fast progress in clinical use of antibody-based immunotherapy, given the superior efficacy commonly achieved in clinic and the limited toxicity. However, personalization of treatment remains of major importance, both to achieve better clinical performance for monotherapy and to identify the best combinations on a patient-by-patient basis. Predicting patient's response is complex due to the need to characterize both tumor response and immunologic mechanisms possibly activated by the therapy, including antibody dependent cellular cytotoxicity (ADCC). We present the Inter-Cell Networking Profiling (ICNP), a novel analytical method enabling a comprehensive and precise characterization of the modulatory effect of immunotherapies on immune-tumor cell interactions. ICNP works on the Open Microwell (OMW) microfluidic system which recreates 20,000 unique cell clusters from ex-vivo patient samples and exposes them to anticancer drugs. We validated the ICNP using multiple myeloma (MM) patient samples to characterize the efficacy of Daratumumab, an anti-CD38 antibody (Ab). Bone marrow samples in EDTA were collected from 11 MM patients. 8 samples were processed by Ficoll-Paque, preserving the original composition of effector (E) and target (T) cells, i.e. NK and plasma cells respectively. 3 samples were processed to obtain co-cultures of WBC depleted of plasma cells (which include NK cells) and U-266 MM cell line. NK and plasma cells were stained with anti-CD16/CD56 and anti-CD138 fluorescently-labeled Abs, respectively. Propidium Iodide (PI) was used as death marker. A statistical model was created to project the optimal experimental setup (E:T ratio, cell concentration) to maximize the co-localization of E/T cells in the 20,000 microwells of the OMW platform. After seeding, cells were incubated with Daratumumab 10µg/mL or no drug and analyzed through fluorescence time lapse microscopy for up to 12 hours. ICNP analysis first separates microwells with both E/T cells in close proximity from those not featuring both cell types (Fig 1A). Then, anti-CD38 efficacy is evaluated in microwells with E/T co-localization, thus implementing a miniaturized ADCC assay (Fig 1B). At the same time, spontaneous NK activity is measured in microwells with E/T co-localization and no drug, while direct effect of the drug on target cells can be measured in microwells with T but not E cells. We first validated our statistical model of co-localization in microwells against the actual number of wells with E/T co-localization (correlation R2: 0.79-0.97, n=5). Then, we characterized the immune composition of 8 MM patients samples with the OMW, finding that E/T cell fractions were in the ranges 5-21% (E) and 1-28% (T). Interestingly, according to our statistical model, co-localization occurs in at least 1% of microwells for all the 8 samples, making ICNP applicable with good statistical significance in the OMW system on ex-vivo clinical samples without any pre-enrichment. Finally, ICNP was evaluated on 4 cases (3 obtained by mixing patient's WBC with U-266 MM cell line and 1 complete MM patient sample). We measured the effect of anti-CD38 Ab using i) the standard approach, considering all microwells regardless of the co-presence of E and T; ii) ICNP approach, considering only microwells featuring E/T co-localization. Results show that drug effect is much evident with ICNP in all the 4 cases with an average increase in target cell death of 40%, indicating a higher sensitivity of this approach than the averaged analysis (Fig 1C, right). Importantly, in one case, the standard analysis did not identify significant differences between anti-CD38 treated and control cells, that could instead be observed with ICNP (p<0.0001, 89% cell death) (Fig 1C, left). These results demonstrate that ICNP can determine the efficacy of the therapy taking into account the fitness of single NK cells that is commonly lost in averaged measurements. ICNP proved to enable a comprehensive profiling of the immune system by evaluating in one test the immune composition and the fitness of immune cells, both native and drug-treated. These results open the opportunity to develop functional precision medicine approaches for predicting patient's response to drugs with immune-mediated mechanisms of action. AB and RR equally contributed Figure 1 Disclosures Bettelli: CellPly.S.r.l.: Employment. Ruggiano:Cellply S.r.l.: Employment. Bocchi:CellPly S.r.l.: Employment. Rocchi:CellPly.S.r.l.: Employment. Faenza:CellPly S.r.l.: Employment. Zamagni:Janssen: Honoraria, Other: Advisory board, Speakers Bureau; Amgen: Honoraria, Other: Advisory board, Speakers Bureau; BMS: Honoraria, Other: Advisory Board, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Sanofi: Honoraria, Other: Advisory Board, Speakers Bureau; Celgene Corporation: Honoraria, Other: Advisory board, Speakers Bureau. Bettelli:CellPly S.r.l.: Employment. Rambelli:CellPly S.r.l.: Employment. Guadagnuolo:CellPly S.r.l.: Employment. Pecorari:CellPly S.r.l.: Employment. Giulianelli:CellPly S.r.l.: Employment. Pisani:CellPly S.r.l.: Employment. Biscarini:CellPly S.r.l.: Employment. Cavo:amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Guerrieri:CellPly S.r.l.: Equity Ownership. Bocchi:CellPly S.r.l.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
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Brand, Marjorie. "Understanding Erythropoiesis Using Quantitative Proteomics and Single Cell Mass Cytometry." Blood 134, Supplement_1 (November 13, 2019): SCI—21—SCI—21. http://dx.doi.org/10.1182/blood-2019-121109.
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The process of erythropoiesis whereby hematopoietic stem cells (HSC) differentiate into cells with increasingly restricted potential is regulated by a network of lineage-specifying transcription factors (LS-TFs) that promote erythropoiesis while simultaneously repressing other hematopoietic lineages. While TFs that stimulate differentiation towards the erythroid lineage (such as GATA1 and KLF1/EKLF) are more abundant in erythroid progenitors and precursors, these proteins are also expressed in hematopoietic stem/progenitor cells (HSPCs) although at very low levels. This suggests that the dosage of TFs plays an important role in lineage determination. Consistent with this, previous lineage reprogramming experiments have shown that ectopic expression of the same LS-TF can give rise to distinct cell fates depending on the TF's abundance. Furthermore, a controversial model proposed that TFs belonging to competing hematopoietic lineages are co-expressed in bipotential progenitors, and that changes in their relative levels drive differentiation towards one fate or another. Taken together, this suggests that changes in LS-TFs stoichiometry may be central to cell fate choice and lineage commitment. While gene regulatory networks have been established to model the process of cell fate decision in bipotential progenitors, these network models are based on mRNA measurements and have not integrated protein levels of TFs. This is a problem as protein levels do not always correlate with mRNA levels, and as such the gene regulatory network underlying erythroid lineage determination is currently unclear. While standard proteomic approaches such as Western blot or data-dependent mass spectrometry (i.e. shotgun mass spectrometry) are useful to measure changes in the relative level of a single protein over time, these approaches do not provide information on the relative levels between several proteins in the same sample, and as such, changes in protein stoichiometry for master regulators of erythropoiesis remain mostly unknown. To address these questions and to provide a better understanding of the role and importance of quantitative changes in LS-TFs for the process of erythroid lineage commitment, we have used a combination of single cell proteomic (i.e. mass cytometry or CyTOF) and targeted mass spectrometry (i.e. SRM for selected reaction monitoring coupled with the spiking of known amounts of internal standard peptides) approaches to measure changes in protein levels of master regulators of hematopoiesis and erythropoiesis. As a model system for human erythropoiesis, we used cord-blood derived CD34+ HSPCs that are driven to differentiate along the erythroid lineage using a combination of growth factors and cytokines 1. Cells were harvested every second day from HSPCs to differentiated erythroid cells. For CyTOF analysis, cells were barcoded at each time-point, combined and stained with a cocktail of antibodies against 11 cell surface markers and 16 TFs. Clustering analysis was then used to establish a temporal trajectory of erythropoiesis. The data revealed that competing LS-TFs proteins (e.g. KLF1 a pro-erythroid factor and FLI1 a pro-megakaryocyte factor) are co-expressed in bipotential progenitors at equimolar levels. Furthermore, relative levels of KLF1 vs FLI1 change gradually as the cells progress along the erythroid trajectory. Finally, ectopic expression of FLI1 in early progenitors actively deviates cells from their preferred erythroid trajectory towards a megakaryocytic lineage 2. Thus, our results support the concept that temporally-regulated quantitative changes in TFs protein levels in individual hematopoietic progenitors are key determinants of cell fate decision in human erythropoiesis. Current studies are ongoing to identify additional pairs of LS-TFs that regulate other hematopoietic transitions. Furthermore, a dynamic gene regulatory network of erythroid lineage commitment that integrates protein and mRNA data for master regulators of hematopoiesis has been established. Giarratana MC, Kobari L, Lapillonne H, et al. Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells. Nat Biotechnol. 2005;23(1):69-74. Palii CG, Cheng Q, Gillespie MA, et al. Single-Cell Proteomics Reveal that Quantitative Changes in Co-expressed Lineage-Specific Transcription Factors Determine Cell Fate. Cell Stem Cell. 2019;24(5):812-820 e815. Disclosures No relevant conflicts of interest to declare.
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Lonergan, T., A. Harvey, J. Zhao, B. Bavister, and C. Brenner. "287 ACTIVE MITOCHONDRIA ARE PRESENT IN MOUSE AND MONKEY EMBRYONIC STEM CELL LINES." Reproduction, Fertility and Development 20, no. 1 (2008): 223. http://dx.doi.org/10.1071/rdv20n1ab287.
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The inner cell mass (ICM) of the blastocyst develops into the fetus after uterine implantation. Prior to implantation, ICM cells synthesize ATP by glycolytic reactions. We now report that cells of the ICM in 3.5-day-old mouse embryos have too few mitochondria to be visualized with either Mitotracker red (active mitochondria) or an antibody against complex I of OXPHOS. By comparison, all of the surrounding trophectoderm cells reveal numerous mitochondria throughout their cytoplasm. It has largely been assumed that embryonic stem (ES) stem cells derived from the ICM also have few mitochondria, and that replication of mitochondria in the ES cells does not begin until they commence differentiation. We further report that mouse E14 ES cells and monkey ORMES 7 ES cells have considerable numbers of active mitochondria when cultured under standard conditions, i.e., 5% CO2 in air. Both the mouse E14 and monkey ES cell lines expressed two markers of undifferentiated cells, Oct-4 and SSEA-4, and monkey ES cells expressed the undifferentiated cell marker Nanog; however, Oct-4 is nonspecific in monkey ES cells because trophectoderm also expresses this marker, unlike in mice. Ninety-nine percent of the E14 cells examined, and 100% of the ORMES 7 cells, have a visible mitochondrial mass when stained with either Mitoracker red or with an antibody against OXPHOS complex I. The ATP content in the mouse E14 cells (4.13 pmoles ATP/cell) is not significantly different (P = 0.76) from that in a mouse fibroblast control (3.75 pmoles ATP/cell). Cells of the monkey ORMES 7 cell line had 61% of the ATP/cell content (7.55 pmoles ATP/cell) compared to the monkey fibroblast control (12.38 pmoles ATP/cell). Both cell lines expressed two proteins believed to indicate competence of mitochondria to replicate: PolG, the polymerase used to replicate the mitochondrial genome, and TFAM, a nuclear-encoded transcription factor reported to regulate several aspects of mitochondrial function. Both proteins were found to co-localize in the mitochondria. We conclude that when the ICMs are isolated from blastocysts and used to establish these two ES cell lines in cell culture, mitochondrial biosynthesis is activated.
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Yao, Yue Ying, Dennis K. Lee, Stephanie Jarvi, Marjan Farshadi, Minzhi Sheng, Sara Mar, Ori Nevo, and Hon S. Leong. "Histological Characterization of Class I HLA Molecules in Whole Umbilical Cord Tissue Towards an Inexhaustible Graft Alternative for Reconstructive Surgery." Bioengineering 10, no. 1 (January 12, 2023): 110. http://dx.doi.org/10.3390/bioengineering10010110.
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Background: Limited graft availability is a constant clinical concern. Hence, the umbilical cord (UC) is an attractive alternative to autologous grafts. The UC is an inexhaustible tissue source, and its removal is harmless and part of standard of care after the birth of the baby. Minimal information exists regarding the immunological profile of a whole UC when it is considered to be used as a tissue graft. We aimed to characterize the localization and levels of class I human leukocyte antigens (HLAs) to understand the allogenicity of the UC. Additionally, HLA-E and HLA-G are putative immunosuppressive antigens that are abundant in placenta, but their profiles in UC whole tissue are unclear. Hypothesis: The UC as a whole expresses a relatively low but ubiquitous level of HLA-ABC and significant levels of HLA-G and HLA-E. Methods: Healthy patients with no known pregnancy-related complications were approached for informed consent. UCs at term and between 12 and 19 weeks were collected to compare HLA profiles by gestational age. Formalin-fixed paraffin-embedded tissues were sectioned to 5 µm and immunohistochemically stained with a pan-HLA-ABC, two HLA-G-specific, or an HLA-E-specific antibody. Results: HLA-ABC was consistently found present in UCs. HLA-ABC was most concentrated in the UC vessel walls and amniotic epithelium but more dispersed in the Wharton’s Jelly. HLA-E had a similar localization pattern to HLA-ABC in whole UC tissues at both gestational ages, but its protein level was lower. HLA-G localization and intensity were poor in all UC tissues analyzed, but additional analyses by Western immunoblot and mass spectrometry revealed a low level of HLA-G in the UC. Conclusion: The UC may address limitations of graft availability. Rather than the presence of HLA-G, the immunosuppressive properties of the UC are more likely due to the abundance of HLA-E and the interaction known to occur between HLA-E and HLA–ABC. The co-localization of HLA-E and HLA-ABC suggests that HLA-E is likely presenting HLA-ABC leader peptides to immune cells, which is known to have a primarily inhibitory effect.
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Ho, Alyssa N., Violet A. Kiesel, Scott P. Connelly, Michael F. Coleman, and Stephen D. Hursting. "Abstract 3010: Targeting metabolic pathways through pharmacological and chemotherapeutic interventions to improve triple-negative breast cancer therapy." Cancer Research 82, no. 12_Supplement (June 15, 2022): 3010. http://dx.doi.org/10.1158/1538-7445.am2022-3010.
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Abstract Triple-negative breast cancers (TNBCs) lack targeted therapies, leaving surgery and systemic chemotherapy as current standard approaches for treatment. However, chemotherapy resistance is a major clinical challenge. Stimulation of receptor tyrosine kinases such as insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF-1R) activates the protein kinase A (PKA), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling cascades. Activation of these pathways is observed in the majority of TNBC tumors and promotes pro-proliferative signaling. Thus IGF-1R/IR are potential therapeutic targets in TNBC, but clinical trials of anti-IGF-1R/IR therapies have consistently shown minimal and often heterogeneous therapeutic responses. Our objective was to determine whether metabolic reprogramming achieved by combination regimens, specifically the IGF-1R/IR inhibitor BMS-754807 and/or treatment with the platinum-based chemotherapeutic agent, carboplatin, promotes autophagy induction and sensitizes cancer cells to autophagy inhibition. Cytotoxicity of BMS-754807, alone or in combination with carboplatin and/or the autophagy inhibitor, hydroxychloroquine, was assessed in murine (E0771, metM-Wntlung, B6C3TAg 2.51) and human (MDA-MB-231, MDA-MB-468) models of TNBC using MTT assays. Analysis of mitochondrial mass was measured in cells stained with MitoTracker Green FM using flow cytometry. Autophagic flux was measured with an mCherry-EGFP-LC3B tandem fluorescent protein. BMS-754807 (10μM) significantly induced 22-51% cytotoxicity in all cell lines tested, with E0771 cells showing the strongest response. Treatment with BMS-754807 (2.5µM) significantly increased mitochondrial mass (31%) compared to untreated cells. In addition, co-treatment with BMS-754807 and carboplatin further suppressed cell viability and regulated phosphorylation of the double-stranded DNA damage response proteins γ;-H2AX and Chk2. Co-treatment with BMS-754807 and carboplatin also altered the GFP/RFP ratio in LC3-expressing cells, suggesting modulation of autophagic flux. Finally, the addition of the autophagy inhibitor hydroxychloroquine further induced cytotoxicity when coupled with BMS-754807 and carboplatin. This work indicates that IGF-1R/IR inhibition remodels metabolism in TNBC cells, potentially synergizing with carboplatin to induce double stranded DNA damage. Moreover, the combination of IGF-1R/IR and carboplatin can further collaborate with autophagy inhibition to strongly suppress TNBC cell growth. We conclude that inhibiting nutrient-sensing metabolic pathways such as IGF-1R/IR in combination with chemotherapy and/or autophagy inhibition warrants additional study as a strategy to improve therapeutic responses in women with TNBC. Citation Format: Alyssa N. Ho, Violet A. Kiesel, Scott P. Connelly, Michael F. Coleman, Stephen D. Hursting. Targeting metabolic pathways through pharmacological and chemotherapeutic interventions to improve triple-negative breast cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3010.
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Matas, C., J. Gadea, and G. Decuadro-Hansen. "89 EVALUATION OF A CUSHIONED CENTRIFUGATION TECHNIQUE FOR PROCESSING BOAR SEMEN FOR FREEZING." Reproduction, Fertility and Development 17, no. 2 (2005): 195. http://dx.doi.org/10.1071/rdv17n2ab89.
Full textAbstract:
Boar semen freezing procedures include the use of centrifugation to concentrate sperm and remove seminal plasma prior to dilution in freezing extender. The centrifugation techniques employed have necessarily been a compromise between the need to recover as many spermatozoa as possible after centrifugation and the damage caused by pelleting the sperm. The use of an inert, dense, and isotonic solution as a cushion in the bottom of the tube leads to the use of higher-speed centrifugation to ensure maximum sperm recovery. However, it is necessary to know the viability and functionality of the samples after the thawing process. The aim of this work was to evaluate the effect of cushion-technique centrifugation on the in vitro sperm viability and the penetrating capacity after thawing. Sperm-rich fractions from five fertile boars were diluted and cooled to 15°C before centrifugation. Two centrifugation regimes were used: 800g for 10 min called the “standard method” (SM) (Westendorf P etal. 1975 Dtsch. Tierzartl Wochenschr. 82, 261–267) and 1000g for 20 min on an iodixanol isotonic solution 60% w/v gradient (Sigma Chemical Co., St. Louis, MO, USA) called the “cushion method” (CM). Spermatozoa were diluted in lactose/egg-yolk extender, cooled to 5°C over 2 h and then frozen with glycerol and Equex by classic methodology (Westendorf P et al. 1975 Dtsch. Tierzartl Wochenschr. 82, 261–267). Frozen sperm samples were thawed in a circulating water bath at 38°C for 30 s. To detect increases in plasma membrane lipid packing disorder and viability, frozen-thawed samples of sperm were stained with merocyanine 540 (M540) and Yo-Pro 1 (Harrison et al. 1996 Mol. Rep. Dev. 45, 378–391) and evaluated by flow cytometry. In vitro penetration ability was assessed using the homologous in vitro penetration (hIVP) test with immature oocytes (Gadea and Matas 2000 Theriogenology 54, 1343–1357). ANOVA analysis revealed that centrifugation by CM showed higher values of intact viable spermatozoa than SM centrifugation (60.21 v. 54.68%, P < 0.05). The in vitro penetration assay showed no differences in penetration rate or mean number of sperm penetrated per oocyte. However, significant boar and interaction effects were found (P < 0.01). These results indicated that different effects of the treatment were found for every boar. In conclusion, the cushioned centrifugation method gives a simple means of processing porcine semen for freezing more efficiently without loss of fertilizing capacity. This work was supported by AGL-2003-03144.
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Golay, Josee, Marzia Leidi, Elisa Gotti, Giuseppe Palumbo, and Martino Introna. "IL-10 Present in the B Lymphoma Microenvironment Drives Macrophages to Efficient Tumour Suppressing Cells with High Phagocytic Activity in Presence of Rituximab." Blood 112, no. 11 (November 16, 2008): 1582. http://dx.doi.org/10.1182/blood.v112.11.1582.1582.
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Abstract Since macrophages have been implicated as major players in the mechanism of action of Rituximab (Mabthera®) in vivo, we have investigated the factors that modulate their tumour cell killing potential in vitro. Human macrophages expressing CD16, CD32 and CD64, were differentiated from CD14+ peripheral blood monocytes by culture for 5–7 days in presence of M-CSF. Binding of rituximab opsonised target cells was measured after 5 minutes incubation of macrophages with CFSE labelled B-CLL cells and FACS analysis. Phagocytosis was quantified after 2 hours at 37°C by microscopic count of stained slide preparations. ADCC was measured by standard release assays. Rituximab induced specific binding of CD20+ target cells to macrophages and this was followed by phagocytosis, but not ADCC. Phagocytosis was maximal at 0.1 μg/ml rituximab and was not significantly affected by CD20 expression levels on target cells. The CD16A polymorphism at amino acid 158 (Val/Phe) that affects IgG binding did not significantly modify the efficacy of phagocytosis at different rituximab doses, possibly due to the role of additional Fcγ receptors. Indeed phagocytosis was blocked by excess human immunoglobulins. Since macrophages can be differentiated to M1 type or M2 type cells with either GM-CSF or M-CSF, respectively, and can be classically activated by pro-inflammatory stimuli (IFNγ + LPS) or undergo alternative activation with cytokines such as IL-4 or IL-10, we have analysed the effect of these different polarisation programs on the phagocytosis mediated by rituximab. Macrophages differentiated in presence of M-CSF showed a 2–3 fold greater phagocytic capacity compared to GM-CSF induced cells. Furthermore addition of IL-10 significantly increased, whereas IL-4 decreased phagocytosis by both M-CSF and GM-CSF differentiated macrophages. LPS/IFNγ had little effect. Expression of CD16, CD32A/C and CD64 correlated with the phagocytic capacity of the different polarised populations, suggesting that M-CSF and IL-10 induced maximal phagocytosis through upregulation of several activating FcγRs. Several B lymphoma cell lines were observed to secrete 400–1300 pg/ml IL-10 in vitro and co-culture of human macrophages with lymphoma supernatant increased significantly their phagocytic capacity. These data suggest that IL-10 produced in the lymphoma microenviroment may activate macrophages to become M2 type cells with high phagocytic capacity. Thus usually tumour promoting M2 type macrophages within the lymphoma would become tumour suppressing in presence of rituximab.
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Decaens, C., J. Nardelli, J. Bara, and P. Burtin. "Biochemical characterization of a rat oncofetal colonic antigen defined by a monoclonal antibody raised against gastric surface epithelium." Biochemical Journal 293, no. 2 (July 15, 1993): 531–36. http://dx.doi.org/10.1042/bj2930531.
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The 660 epitope was defined by a monoclonal antibody raised against rat gastric surface epithelium scrapings. This epitope, a marker of goblet cell differentiation, shows oncofetal behaviour in the colonic mucosa. We found that it co-purified with gastric mucin glycoproteins. We isolated rat gastric mucus glycoproteins using standard techniques: gastric scrapings in PBS were submitted to isopycnic density gradient centrifugation in CsCl in the presence of proteinase inhibitors. Fractions of relative density 1.4-1.45 with a high neutral sugar/protein ratio were chromatographed on an Ultrogel A4 column. According to the usual criteria, the high-molecular mass glycoproteins recovered in the excluded volume were purified mucins; when stained with periodic acid/Schiff reagent, they showed little migration on 4-15% gradient gel acrylamide electrophoresis. Serine+threonine+proline residues accounted for 35% of the total amino acids; the carbohydrate composition consisted of galactose, fucose, N-acetylgalactosamine and N-acetylglucosamine. These mucus glycoproteins carried the 660 epitope. After disulphide bond reduction, the remaining high-molecular-mass subunits were retained by the Ultrogel A4 column; amino acid and saccharide compositions were generally similar to those of the unreduced fraction. Trypsin digestion of the 660 epitope glycoprotein carrier did not modify its chromatographic and electrophoretic patterns, nor its chemical composition. The 660 epitope was still present after these treatments. However, trypsin digestion of subunits gave rise to smaller components that were retained by an Ultrogel A4 column. The saccharide composition of these fragments was unchanged, but the proportion of serine+threonine+proline residues rose to 46% of the total. These digested subunits had lost nearly all reactivity with monoclonal antibody 660. Our results fit well with the macromolecular model of Carlstedt, Lindgren and Sheehan [(1983) Biochem. J. 213, 427-435]: mucin glycoproteins are homopolymers of subunits assembled end-to-end via disulphide bonds into very large linear macromolecules. After disulphide bond reduction, proteolytic attack sites are uncovered and trypsin digestion results in glycopeptides bearing the typical oligosaccharidic units and with enhanced amounts of serine, threonine and proline, the characteristic amino acids of this hyperglycosylated region of the peptide core. These digested subunits have lost virtually all 660 epitope reactivity. We thus show that the 660 epitope, a determinant of a mucin molecule, is probably associated with the peptide core of the glycoprotein.
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Giorgetti, Giulia, Pamela Becherini, Elena Maroto-Martin, Daniela Fenoglio, Debora Soncini, Claudia Martinuzzi, Santina Bruzzone, et al. "Higher CD56 Expression on Multiple Myeloma Cells Increases CD38 Expression, Reduces Intracellular NAD+ Levels, and Enhances the Efficacy of Daratumumab-Based Treatment Strategies." Blood 142, Supplement 1 (November 28, 2023): 1951. http://dx.doi.org/10.1182/blood-2023-189439.
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NCAM1 (Neural Cell Adhesion Molecule 1), also known as CD56, is a member of the immunoglobulin superfamily and is deregulated in many tumors. In multiple myeloma (MM), high levels of CD56 are observed on MM cells in 70% of patients. This molecule has several functions including tumor growth, adhesion and response to therapy, as suggested by several studies. However, an extensive analysis of the specific landscape underlying its presence and its role as a prognostic biomarker is lacking, especially in MM. Here, we evaluated CD56 surface levels on tumor cells collected from 136 newly diagnosed MM patients (Policlinic San Martino Hospital, Genoa, Italy) and found that low CD56 expression significantly correlated with extramedullary disease (EMD) (p = .016) while high CD56 levels were associated with a lower risk of disease progression or death (p = .003). When evaluating the surface expression of MM cells, we surprisingly found a positive correlation between CD56 and CD38 levels (p < .0001), which was replicated in a panel of MM cell lines (p = .02). To gain insight into the biological significance of this, we challenged a panel of MM cell lines with Daratumumab (DARA), a standard-of-care CD38 antibody that kills MM cells through multiple mechanisms including antibody-dependent cellular cytotoxicity (ADCC). Given the correlation between the expression of CD56 and CD38, we hypothesized that higher CD56 expression would lead to enhanced DARA-mediated ADCC. We performed standard 3-hour ADCC assays using NK cells from healthy donors as effector cells and CD56-positive or -negative cell lines. Myeloma cells were stained with calcein-AM and co-cultured with NK cells at indicated effector:target (E:T) ratios. The assay was assessed in the presence or absence of 1μg/mL Daratumumab. We found that MM cell lines with high expression of CD56 were more sensitive to DARA-mediated NK cell killing (p = .004 at ratio 1:1 E:T). Consistently, MM patients with higher levels of CD56 (N = 8) had better overall response rates to DARA-based therapies compared to patients with lower levels (N = 9). CD56 and CD38 are not known to interact, so this correlation may be mediated by the NAD+ biosynthesis pathway. CD38 is a critical enzyme for breaking down NAD + in cells. We found that ectopic expression of CD56 led to lower intracellular NAD + levels (MM1S overexpressing CD56 p = .0875; RPMI 8226 overexpressing CD56 p = .0003) and increased CD38 enzymatic activity (MM1S overexpressing CD56 p = .0035; RPMI 8226 overexpressing CD56 p = .0478), as measured by a cyclase activity assay. As a result, CD56 surface levels affected MM cells' sensitivity to NAD +-lowering agents, including next-generation NAMPT inhibitors. Indeed, MTS assays confirm that CD56-overexpressing MM cells were more vulnerable to these small molecules, whereas CD56 silencing made cells more resistant. In conclusion, our data suggest CD56 levels as an important determinant of sensitivity to DARA-based therapies. Moreover, our results suggest the next-generation NAMPT inhibitors as an additional therapeutic strategy for CD56-expressing MM patients.