Human Aspirates Research Articles

Enzymatic prodrug degradation in the fasted and fed small intestine: In vitro studies and interindividual variability in human aspirates

The aim of the current study was (1) to develop an automation-based protocol for in vitro assessment of enzymatic drug stability at fasted- and fed-state intestinal conditions, (2) to characterize the inter-individual variability of drug degradation in fasted- and fed-state human intestinal fluids, and (3) to compare the obtained in vitro results to drug degradation in human intestinal fluids by taking variability into account. In human intestinal fluids, drug degradation displayed large inter-individual variability, with coefficients of variance generally ranging between 30 and 70 %. The effect of food on the inter-individual variability was highly dependent on the type of drug. The increase of pH in the range between 5.0 and 7.0 significantly accelerated the degradation rate of the studied drugs both in the in vitro and ex vivo experiments. In contrast, the increase of bile salt and phospholipid concentrations in the in vitro screen decreased strongly the degradation rate of the hydrophobic drugs. The developed automated in vitro screen mimicked relatively well the ex vivo degradation of all drugs in the fasted state, whereas in the fed state the degradation of only one of the drugs was adequately reproduced.

Exploiting the Role of Hypoxia-Inducible Factor 1 and Pseudohypoxia in the Myelodysplastic Syndrome Pathophysiology.

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem (HSCs) and/or progenitor cells disorders. The established dependence of MDS progenitors on the hypoxic bone marrow (BM) microenvironment turned scientific interests to the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 facilitates quiescence maintenance and regulates differentiation by manipulating HSCs metabolism, being thus an appealing research target. Therefore, we examine the aberrant HIF-1 stabilization in BMs from MDS patients and controls (CTRLs). Using a nitroimidazole–indocyanine conjugate, we show that HIF-1 aberrant expression and transcription activity is oxygen independent, establishing the phenomenon of pseudohypoxia in MDS BM. Next, we examine mitochondrial quality and quantity along with levels of autophagy in the differentiating myeloid lineage isolated from fresh BM MDS and CTRL aspirates given that both phenomena are HIF-1 dependent. We show that the mitophagy of abnormal mitochondria and autophagic death are prominently featured in the MDS myeloid lineage, their severity increasing with intra-BM blast counts. Finally, we use in vitro cultured CD34+ HSCs isolated from fresh human BM aspirates to manipulate HIF-1 expression and examine its potential as a therapeutic target. We find that despite being cultured under 21% FiO2, HIF-1 remained aberrantly stable in all MDS cultures. Inhibition of the HIF-1α subunit had a variable beneficial effect in all <5%-intra-BM blasts-MDS, while it had no effect in CTRLs or in ≥5%-intra-BM blasts-MDS that uniformly died within 3 days of culture. We conclude that HIF-1 and pseudohypoxia are prominently featured in MDS pathobiology, and their manipulation has some potential in the therapeutics of benign MDS.

In vitro megakaryocyte culture from human bone marrow aspirates as a research and diagnostic tool

Megakaryocytes (MKs) are relatively rare in bone marrow, comprising <0.05% of the nucleated cells, which makes direct isolation from human bone marrow impractical. As such, in vitro expansion of primary MKs from patient samples offers exciting fundamental and clinical opportunities. As most of the developed ex vivo methods require a substantial volume of biomaterial, they are not widely performed on young patients. Here we propose a simple, robust, and adapted method of primary human MK culture from 1 mL of bone marrow aspirate. Our technique uses a small volume of bone marrow per culture, uses straightforward isolation methods, and generates approximately 6 × 105 mature MKs per culture. The relative high cell purity and yield achieved by this technique, combined with efficient use of low volumes of bone marrow, make this approach suitable for diagnostic and basic research of human megakaryopoiesis.

Human MDSCs derived from the bone marrow maintain their functional ability but have a reduced frequency of induction in the elderly compared to pediatric donors

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immunosuppressive cells developing from myeloid progenitors, which are enriched in pathological conditions such as cancer, and are known to inhibit the functions of effector T cells. During aging, several changes occur both at the adaptive and innate immune system level, in a process defined as immunoscenescence. In particular, the low-grade inflammation state observed in the elderly appears to affect hematopoiesis. We previously demonstrated that the combination of GM-CSF and G-CSF drives the in vitro generation of bone marrow-derived MDSCs (BM-MDSCs) from precursors present in human bone marrow aspirates of healthy donors, and that these cells are endowed with a strong immune suppressive ability, resembling that of cancer-associated MDSCs. In the present work we investigated BM-MDSCs induction and functional ability in a cohort of pediatric versus elderly donors. To this aim, we analyzed the differences in maturation stages and ability to suppress T cell proliferation. We found that the ex vivo distribution of myeloid progenitors is similar between pediatric and elderly individuals, whereas after cytokine treatment a significant reduction in the more immature compartment is observed in the elderly. Despite the decreased frequency, BM-MDSCs maintain their suppressive capacity in aged donors. Taken together, these results indicate that in vitro induction of MDSCs from the BM is reduced with aging and opens new hypotheses on the role of age-related processes in myelopoiesis.

Abstract 4499: Cancer targets early B-cell precursors to generate metastasis-promoting Bregs by promoting their premature emigration from the bone marrow and expansion in the circulation

Abstract We previously reported that breast cancer (BC) generates metastasis-promoting regulatory B cells (CD25+ tumor-evoked Bregs). Because phenotypically these cells resembled bone marrow (BM) Rag+ B-cell precursors, such as large Pre-B cells, we hypothesized whether cancer uses them to generate Bregs. To investigate this idea, we used mouse and human BM aspirates, human PBMCs from BC patients, and mice with different orthotopic cancers, such as highly metastatic 4T1 breast cancer cells and EMT6. We report that breast cancer indeed uses BM B-cell precursors to generate Bregs. However, to do this, cancer expresses thymic stromal lymphopoietin (TSLP). Mechanistically, cancer-produced TSLP downregulates surface expression of CXCR4 and α4β1 (VLA-4) of B-cell precursors, thereby impairing their BM retention and increasing their exit into the circulation. TSLP also enables survival and proliferation of these BM emigrants, while other factors such as 5-lipoxygenase metabolites convert them into Bregs. The loss of TSLP expression in cancer cells alone or TSLPR deficiency in B cells not only reduce pre-B cells in the circulation, but also retard lung metastasis. Overall, our data reveal a previously unknown function of cancer-produced TSLP as inducers of premature B-cell precursor emigration from BM to support Breg generation. The results also suggest that the TSLP-pre-B cell axis can be an attractive therapeutic target. Citation Format: Emeline Ragonnaud, Kanako Moritoh, Monica Bodogai, Soizic Garaud, Chen Chen, Xin Wang, Karen Willard-Gallo, Arya Biragyn. Cancer targets early B-cell precursors to generate metastasis-promoting Bregs by promoting their premature emigration from the bone marrow and expansion in the circulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4499.

Transplanting of Adipose Derived Stem Cells Up-Regulate CD4+CD25+Foxp3+ T Cells in a Murine Model of Passive Chronic Immune Thrombocytopenia

Background: Immune thrombocytopenia (ITP) is an autoimmune disorder in which antibodies and/or T cells lead to enhanced peripheral platelet destruction and reduced bone marrow platelet production. The dysregulated CD4+CD25+Foxp3+ T regulatory cells (Tregs) play a critical role in the pathophysiology of ITP. Human adipose-derived mesenchymal stem cells (hADSCs) were recently found to suppress effector T cell responses and to have beneficial effects in various immune disorders. However, it is not clear how ADSCs could affect ITP. This study is aimed to investigate the therapeutic effect of ADSCs on the treatment of ITP mice and explore the underlying mechanisms. Methods: A passive immune thrombocytopenia animal model was induced by injection of the monoclonal antiplatelet antibody MWReg30, and ADSC injection was performed on the 2nd day after ITP induction. PLT counts have been measured by an automatic blood cell counter daily after ADSC transplant for 6 days. The frequency of Tregs in the blood was analyzed by flow cytometry. mRNAs and proteins of Foxp3, TGF-β1 and IL-10 in the spleen, thymus and lymph nodes were detected by RT-PCR and western blotting respectively. Findings: Transplant of ADSCs on ITP mice led to increased PLT counts, an up-regulation of Tregs in the blood, and an up-regulation of Foxp3, TGF-β1 and IL-10 mRNA and protein in the spleen, thymus and lymph nodes as compared with the untreated ITP mice. Conclusion: ADSCs transplant may be an available method in therapy of ITP. ADSCs transplant may affect proliferation and function of Tregs in ITP, and Foxp3, TGF-β1 and IL-10 may be involved in this process. Funding: This work was supported by the Youth Program of National Natural Science Founds (8150091), The Doctoral Start Project of Natural Science Foundation of Guangdong Province(2015A030310022) and the Youth Program of Natural Science Founds of Hunan Province (2018JJ3472). Declaration of Interest: The authors declare that they have no conflict of interest. Ethical Approval: All experimental procedures conformed to the Guidelines of Animal Experiments from the Chinese Committee of Medical Ethics and were approved by the Commission of Animal Research, Sun Yat-sen University Medical Center. Raw human aspirates from patients undergoing lipectomy were collected after obtaining informed consent according to procedures approved by the Ethics Committee of the Chinese Academy of Medical Sciences and the First Affiliated Hospital of South China University.

Biomimicry of glycosaminoglycans in the bone marrow microenvironment favour the expansion of highly potent human mesenchymal stem cells

Background & Aim Mesenchymal stem cell (MSC) therapy offers significant potential for musculoskeletal regeneration following trauma or disease. However, the low abundance of MSCs in human bone marrow aspirates necessitates further expansion in culture before their clinical application. Such ex vivo expansion can lead to a loss of stem cell characteristics, and thus unpredictable therapeutic outcomes. This has necessitated moves to develop strategies that lead to retention of MSC potency over serial passages. Studies of the MSC secretome have highlighted the importance of endogenous FGF2 for their survival and maintenance of stemness. In this study, we aimed to establish a glycosaminoglycan-rich culture microenvironment that would bind and activate endogenous FGF2 to increase the growth of multipotent MSCs suitable for therapeutic application. Methods, Results & Conclusion We show that MSC potency and therapeutic efficacy can be enhanced by sustaining endogenous FGF2 signalling through supplementation with an affinity-selected heparan sulfate co-factor (termed HS8) generated by peptide-based affinity chromatography. Also, HS8 improves the bioavailability of FGF2, so providing MSCs access to active FGF2. This results in a significant increase in the pool of MSCs and sustained growth of multipotent MSCs over extended passaging. Importantly, this increase in cell division does not accelerate telomere loss, or adversely affect colony-forming ability, cell surface marker expression or multipotentiality. When used to treat osteochondral injury to the femoral trochlear groove of NIH nude rats, improvements in ICRS II and O'Driscoll scores were observed, with > 70% of defects treated with MSCs supplemented with HS8 achieving high scores compared with ∼ 50% for control. In a follow-on micropig osteochondral defect study, treatment with MSCs supplemented with HS8 showed significant increases in ICRS II and O'Driscoll scores compared to control. Moreover, Magnetic Resonance Imaging (MRI) analysis demonstrated reduced osteochondral lesioning following treatment with MSCs supplemented with HS8, with Instron biomechanical testing highlighting an associated improvement in biomechanical properties over control. Collectively, these data show that modulation of endogenous FGF2 signals by select heparan glycosaminoglycans represents an effective strategy for the bioprocessing of human mesenchymal stem cells with sustained therapeutic potency.

Detection of DNA in Human Blastocyst Cavity Aspirate by Multiplex PCR

Preimplantation genetic testing (PGT) is a modern method of detection of chromosomal and genetic abnormalities in a human embryo before its transfer to the uterus. The genetic material is obtained by embryo biopsy. Here, we attempted to evaluate the efficiency of a method of noninvasive biopsy—aspiration of the blastocoel contents (blastocentesis) of human embryos. In this study, a biopsy was carried out human embryos with low morphological characteristics (3–4 CC according to Gardner grading) on day 6–7 of development. DNA obtained from the aspirate, as well as from the blastocyst, was analyzed by QF-PCR (quantitative fluorescent polymerase chain reaction) after whole genome amplification. In total, 24 blastocysts and aspirate samples obtained from them were analyzed; assayable DNA was found in seven (29%) aspirate samples from the blastocyst cavity, and this DNA was identical to blastocyst DNA in five (71%) cases. Thus, it was shown that, using aspiration of the blastocoel fluid of the human embryo, it is possible to obtain DNA suitable for analysis by molecular genetic methods. The features and advantages of the use of multiplex QF-PCR method combined with whole genome amplification for studying DNA obtained during aspiration of the blastocoel fluid are discussed. The prospects of DNA obtainment by the noninvasive biopsy method for preimplantation genetic testing (PGT) in the routine practice of infertility treatment and prevention of chromosomal and genetic abnormalities in newborns are considered.

Physiologically Based Absorption Modeling of Salts of Weak Bases Based on Data in Hypochlorhydric and Achlorhydric Biorelevant Media.

Physiologically based absorption modeling has been attracting increased attention to study the interactions of weakly basic drug compounds with acid-reducing agents like proton-pump inhibitors and H2 blockers. Recently, standardized gastric and intestinal biorelevant media to simulate the achlorhydric and hypochlorhydric stomach were proposed and solubility and dissolution data for two model compounds were generated. In the current manuscript, for the first time, we report the utility of these recently proposed biorelevant media as input into physiologically based absorption modeling. Where needed, data collected with the biorelevant gastrointestinal transfer (BioGIT) system were used for informing the simulations in regard to the precipitation kinetics. Using two model compounds, a HCl salt and a semi-fumarate co-crystal which as expected dissolve to a greater extent in these media (and in gastric and intestinal human aspirates) compared to what the pH-solubility profile of the free form would suggest, we demonstrate successful description of the plasma concentration profiles and correctly predicted the lack of significant interaction after administration with pantoprazole or famotidine, respectively. Thus, the data reported in this manuscript represent an initial step towards defining biorelevant input for such simulations on interactions with acid-reducing agents.

Impact of mechanical stimulation on the chondrogenic processes in human bone marrow aspirates modified to overexpress sox9 via rAAV vectors

BackgroundEvaluation of gene-based approaches to target human bone marrow aspirates in conditions of mechanical stimulation that aim at reproducing the natural joint environment may allow to develop improved treatments for articular cartilage injuries. In the present study, we investigated the potential of rAAV-mediated sox9 gene transfer to enhance the chondrogenic differentiation processes in human bone marrow aspirates under established hydrodynamic conditions compared with the more commonly employed static culture conditions.MethodsFresh human bone marrow aspirates were transduced with rAAV-FLAG-hsox9 (40 μl) and maintained for up to 28 days in chondrogenic medium under mechanically-induced conditions in dynamic flow rotating bioreactors that permit tissue growth and matrix deposition relative to static culture conditions. The samples were then processed to examine the potential effects of sox9 overexpression on the cellular activities (matrix synthesis, proliferation) and on the chondrogenic differentiation potency compared with control treatments (absence of rAAV vector; reporter rAAV-lacZ, rAAV-RFP, and rAAV-luc gene transfer).ResultsProlonged, significant sox9 overexpression via rAAV was achieved in the aspirates for at least 28 days when applying the rAAV-FLAG-hsox9 construct, leading to higher, prolonged levels of matrix biosynthesis and to enhanced chondrogenic activities relative to control treatments especially when maintaining the samples under mechanical stimulation. Administration of sox9 however did not impact the indices of proliferation in the aspirates. Remarkably, sox9 gene transfer also durably delayed hypertrophic and osteogenic differentiation in the samples regardless of the conditions of culture applied versus control treatments.ConclusionsThe current observations show the value of genetically modifying human bone marrow aspirates upon mechanical stimulation by rAAV sox9 as a promising strategy for future treatments to improve cartilage repair by implantation in lesions where the tissue is submitted to natural mechanical forces.

The impact of reduced gastric acid secretion on dissolution of salts of weak bases in the fasted upper gastrointestinal lumen: Data in biorelevant media and in human aspirates

PurposeTo propose media for simulating the intragastric environment under reduced gastric acid secretion in the fasted state at three levels of simulation of the gastric environment and evaluate their usefulness in evaluating the intragastric dissolution of salts of weak bases. To evaluate the importance of bicarbonate buffer in biorelevant in vitro dissolution testing when using Level II biorelevant media simulating the environment in the fasted upper small intestine, regardless of gastric acid secretions. MethodsMedia for simulating the hypochlorhydric and achlorhydric conditions in stomach were proposed using phosphates, maleates and bicarbonates buffers. The impact of bicarbonates in Level II biorelevant media simulating the environment in upper small intestine was evaluated so that pH and bulk buffer capacity were maintained. Dissolution data were collected using two model compounds, pioglitazone hydrochloride and semifumarate cocrystal of Compound B, and the mini-paddle dissolution apparatus in biorelevant media and in human aspirates. ResultsSimulated gastric fluids proposed in this study were in line with pH, buffer capacity, pepsin content, total bile salt/lecithin content and osmolality of the fasted stomach under partial and under complete inhibition of gastric acid secretion. Fluids simulating the conditions under partial inhibition of acid secretion were useful in simulating concentrations of both model compounds in gastric aspirates. Bicarbonates in Level III biorelevant gastric media and in Level II biorelevant media simulating the composition in the upper intestinal lumen did not improve simulation of concentrations in human aspirates. ConclusionsLevel III biorelevant media for simulating the intragastric environment under hypochlorhydric conditions were proposed and their usefulness in the evaluation of concentrations of two model salts of weak bases in gastric aspirates was shown. Level II biorelevant media for simulating the environment in upper intestinal lumen led to underestimation of concentrations in aspirates, even when bicarbonate buffer was used.

In Vitro Assessment of Antifungal Caspofungin on Leishmania donovani Culture Isolation.

Leishmania parasite isolation from the human aspirates is always challenging due to most probability of the fungal contamination and the use of antifungal drug which could support the selective growth of the Leishmania parasite. In this study, we examine the effect of antifungal drug caspofungin on the promastigote stage of Leishmania donovani. Promastigote parasite was cultivated in M199 + 20% heat-inactivated fetal calf serum and plated in 96-well plates. Seven different concentrations of caspofungin (512 µg/ml to 8 µg/ml) were exposed to parasites, and 50% inhibitory concentration (IC50) was calculated. Candida spp. was used in the experiments to know the efficacy of caspofungin to inhibit fungal growth. The IC50 values of Leishmania strains ranged from 23.02 to 155.80 µg/ml (mean 90.25 ± 39.01 µg/ml), and it was significantly higher (P value = 0.02) than IC50 values of Candida spp. (ranged from 0.001 to 0.12 µg/ml, mean = 0.05 ± 0.05 µg/ml). The reduced growth rate of the parasite was found with exposure to 50 µg/ml of caspofungin. Growth inhibition of Leishmania donovani is significantly lower with caspofungin and could be used to protect the parasite cultivation from fungal contamination.

Evaluation of the Impact of Excipients and an Albendazole Salt on Albendazole Concentrations in Upper Small Intestine Using an In Vitro Biorelevant Gastrointestinal Transfer (BioGIT) System

An in vitro biorelevant gastrointestinal transfer (BioGIT) system was assessed for its ability to mimic recently reported albendazole concentrations in human upper small intestine after administration of free base suspensions to fasted adults in absence and in presence of supersaturation promoting excipients (hydroxypropylmethylcellulose and lipid self-emulsifying vehicles). The in vitro method was also used to evaluate the likely impact of using the sulfate salt on albendazole concentrations in upper small intestine. In addition, BioGIT data were compared with equilibrium solubility data of the salt and the free base in human aspirates and biorelevant media. The BioGIT system adequately simulated the average albendazole gastrointestinal transfer process and concentrations in upper small intestine after administration of the free base suspensions to fasted adults. However, the degree of supersaturation observed in the duodenal compartment was greater than in vivo. Albendazole sulfate resulted in minimal increase of albendazole concentrations in the duodenal compartment of the BioGIT, despite improved equilibrium solubility observed in human aspirates and biorelevant media, indicating that the use of a salt is unlikely to lead to any significant oral absorption advantage for albendazole.

Bile Salt Micelles and Phospholipid Vesicles Present in Simulated and Human Intestinal Fluids: Structural Analysis by Flow Field–Flow Fractionation/Multiangle Laser Light Scattering

Knowledge about colloidal assemblies present in human intestinal fluids (HIFs), such as bile salt micelles and phospholipid vesicles, is regarded of importance for a better understanding of the in vivo dissolution and absorption behavior of poorly soluble drugs (Biopharmaceutics Classification System class II/IV drugs) because of their drug-solubilizing ability. The characterization of these potential drug-solubilizing compartments is a prerequisite for further studies of the mechanistic interplays between drug molecules and colloidal structures within HIFs. The aim of the present study was to apply asymmetrical flow field–flow fractionation (AF4) in combination with multiangle laser light scattering in an attempt to reveal coexistence of colloidal particles in both artificial and aspirated HIFs and to determine their sizes. Asymmetrical flow field–flow fractionation/multiangle laser light scattering analysis of the colloidal phase of intestinal fluids allowed for a detailed insight into the whole spectrum of submicron- to micrometer-sized particles. With respect to the simulated intestinal fluids mimicking fasted and fed state (FaSSIF-V1 and FeSSIF-V1, respectively), FaSSIF contained one distinct size fraction of colloidal assemblies, whereas FeSSIF contained 2 fractions of colloidal species with significantly different sizes. These size fractions likely represent (1) mixed taurocholate-phospholipid-micelles, as indicated by a size range up to 70 nm (in diameter) and a strong UV absorption and (2) small phospholipid vesicles of 90-210 nm diameter. In contrast, within the colloidal phase of the fasted state aspirate of a human volunteer, 4 different size fractions were separated from each other in a consistent and reproducible manner. The 2 fractions containing large particles showed mean sizes of approximately 50 and 200 nm, respectively (intensity-weighted mean diameter, Dz), likely representing mixed cholate/phospholipid micelles and phospholipid vesicles, respectively. The sizes of the smaller 2 fractions being below the size range of multiangle laser light scattering analysis (<20 nm) and their strong UV absorption indicates that they represent either pure cholate micelles or small mixed micelles. Within the colloidal fraction of the fed-state human aspirate, similar colloidal assemblies were detected as in the fasted state human aspirates. The observed differences between SIF and HIF indicate that the simulated intestinal fluids (FaSSIF-V1 and FeSSIF-V1) represent rather simplified models of the real human intestinal environment in terms of coexisting colloidal particles. It is hypothesized that the different supramolecular assemblies detected differ in their lipid composition, which may affect their affinity toward drug compounds and thus the drug-solubilizing capabilities.

Standardized quantification of human bone marrow mesenchymal stromal cells based on a flow cytometric assay

Conclusion The mesenchymal stromal cell (MSC) content is a major quality determinant of human bone marrow aspirates (BMA). It depends on the aspiration site, contamination with peripheral blood, as well as the aspiration procedure, and it negatively correlates with donor age. The common way of quantifying MSCs is based on the cultivation of cells using the colony forming unit– fibroblast (CFU-F) assay, which is time consuming and highly variable. Cuthbert et al. described a close linear relationship between the number of CFU-F colonies counted manually after 14 days of culture and the number of CD271bright cells per mL of BMA1. The aim of this work was the optimization of a fast and reproducible flow cytometry–based MSC quantification method enabling prospective quality assessment of BMA. In previous experiments we have shown that only CD271bright cells, which also express MSCA-1, give rise to CFU-F. MSCs were isolated from 2×107 bone marrow mononuclear cells (BM MNC) using CD271 (LNGFR)-APC and Anti-APC MicroBeads. Samples of all fractions were labeled with CD45-FITC. PI fluorescence and light scatter signals were used for gating live cells. The positive fraction contained CD271+ cells with a purity of about 86% (Mean: 83±4.1%; n = 4). This population contained about 73% CD271CD45 cells (A, orange fraction) and about 13% CD271CD45 cells (A, red fraction). Further separation of CD271 and CD271 cells was achieved by flow sorting. The different fractions were cultivated and CFU-F numbers were counted after 14 days. The numbers of CFU-F were about 40-fold higher when MSCs were cultivated from the CD271/ CD45 fraction compared to MSCs obtained by plastic adherence (PA). No CFU-Fs were detected in the CD271– and CD271CD45 cell fractions (A). Counterstaining of isolated CD271+ cells revealed a 100% co-expression of MSCA-1 in CD271CD45 cells (B). CD271CD45 MSCs met ISCT criteria2 after culture expansion (3 passages) with respect to MSCspecific marker expression (C) and their differentiation potential towards osteocytes, adipocytes, and chondrocytes (D, left to right)

Quantitative Determination of Cerberin in Seed Extract of Cerbera odollam and Rat Serum by High Performance Thin Layer Chromatography

Cerberin (2-o-Acetyl neriifolin) is the principal cardiac glycoside present in the seeds of Cerbera odollam (Fig 1) belonging to Apocynacea family. The seeds of Cerbera odollam are used as a poison for suicidal as well as homicidal purpose by people around the world. Its detection in the body fluids is somewhat difficult.The present study aims to develop a HPTLC method to identify and quantify Cerberin present in the seeds of Cerbera odollam and Cerberin present in Rat Serum. Chloroform and methanol in the ratio of 9.6:0.4 v/v was employed as mobile phase for Cerberin. Linear ascending development was carried out in a 10 x 10 cm twin trough glass chamber (CAMAG) saturated with the mobile phase. Detection and quantitation was performed by densitometric scanning at 254 nm, using deuterium lamp. The proposed HPTLC method provided a good resolution of Cerberin in ethylacetate/ethanol extract of seeds of Cerbera odollam and Cerberin present in rat serum, confirmed by overlaying UV absorption spectra with standard cerberin marker. The method found to be rapid, simple and precise and may be employed for the detection and quantification of cerberin in human serum, aspirates and other body fluids.

Abstract 4855: Proteomic characterization of the extracellular matrix in multiple myeloma

Abstract Background: The extracellular matrix is a major component of the tumor microenvironment, contributing to the regulation of cell survival, proliferation, differentiation and metastasis. In multiple myeloma (MM) ECM components, such as integrins, fibronectin and collagens, have been shown are critical to the pathogenesis of MM and the development of drug resistance. To date, despite some knowledge of the composition of the ECM in tumors, detailed profiling of the composition of the ECM in MM has not been carried out. Recent advances in proteomics have led to the characterization of the ECM and ECM-related proteins (“matrisome”) in normal human tissues and tumors in a systematic and comprehensive approach. Methods: Tumor Xenograft models; MM1S-GFP-Luc+ cells (5x106) were injected intravenously into SCID-Bg mice (n=4/group) and animals underwent weekly bioluminescent imaging (BLI). Mice were sacrificed after two weeks in order to mimic early tumor development (luminescence= 1x105 p/sec/cm2/sr). and compared to mice demonstrating high tumor burden (1x108 p/sec/cm2/sr) 5 weeks post injection. Human bone marrow aspirates; Whole bone marrow was obtained from MM patients (newly diagnosed n=9, relapsed n=9) and healthy human donors (n=9) following written informed consent. Sequential extractions of whole pooled bone marrow from mice and individual bone marrow humans was performed using the CNMCS(Cytosol/Nucleus/Membrane/Cytoskeleton) Compartmental Protein Extraction Kit (Cytomol, CA). Following this, proteins underwent reversed-phase high performance liquid chromatography followed by tandem mass spectrometry (MS). Identified peptide spectra were counted as a semi quantitative measure of abundance. Results: We detected a total of 1202, 982 and 329 unique proteins from enriched whole bone marrow samples from relapsed patients, newly diagnosed patients and mice, respectively. Of these, critical ECM components such as laminins, matrix metalloproteinases and collagens were found to be enriched in human MM ECM in comparison to healthy donors with increased abundance apparent with disease progression in mice. Specifically Bone Marrow Proteoglycan and Proteoglycan 3 were amongst the ECM proteins significantly enriched in the ECM of newly diagnosed patients. PRG3 is a p53 responsive gene that has been demonstrated to be upregulated in apoptotic cells in several cancers, including CLL. The ECM protein Elastin, the expression of which is closely associated with the invasive/metastatic potential of various cancer types, was also upregulated in the MM ECM where it may play an important role in the tumor-ECM interaction. Conclusions: We have profiled the ECM in MM using mass spectrometry with a view to determining the specific components that may be important in MM disease biology. Through this approach molecular mechanisms that influence MM development and progression can be uncovered and potential targets for therapy identified. Citation Format: Siobhan V. Glavey, Alexandra Naba, Manuela Gambella, Alberto Rocci, Antonio Sacco, John Asara, Antonio Palumbo, Richard O. Hynes, Aldo M. Roccaro, Irene M. Ghobrial. Proteomic characterization of the extracellular matrix in multiple myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4855. doi:10.1158/1538-7445.AM2014-4855

In vitro vs. canine data for assessing early exposure of doxazosin base and its mesylate salt

In this study, we evaluated the usefulness of biorelevant in vitro data and of canine data in forecasting early exposure after the administration of two phases of a BCS Class II compound, i.e., doxazosin base (DB) and its mesylate salt (DM). DB, DM, and doxazosin hydrochloride (DH) were prepared and characterized. In vitro data were collected in various media, including human aspirates. Solubilities of DB and DM in human gastric fluid were forecasted by data in fasted state simulating gastric fluid containing physiological components (FaSSGF-V2) but not by data in HClpH 1.8. Unlike data in FaSSGF-V2, dissolution of DB and DM tablets in HClpH 1.6 is rapid. Dissolution of DB tablet in FaSSGF-V2 is incomplete and conversion to DH seems to occur. Differences between DB and DM in dissolution in the small intestine are overestimated in the absence of physiological solubilizers. Using the in vitro data and previously described modeling procedures, the cumulative doxazosin profile in plasma was simulated and the 0–2h profile was used for evaluating early exposure. Individual cumulative doxazosin profiles in plasma, after single DM tablet administrations to 24 adults, were constructed from corresponding actual plasma profiles. Compared with in vitro DM data in pure aqueous buffers, DM data in biorelevant media led to better prediction of early exposure. Based on intersubject variability in early exposure after DM administration and simulated profiles, the administered phase, DB or DM, does not have a significant impact on early exposure. Partial AUCs were used for evaluating early exposure after DB and DM administration in 4 dogs. Early exposure was significantly higher after administration of DM to dogs. Dogs are not appropriate for evaluating differences in early exposure after DB and DM administrations.

The cellular plasticity of human adipocytes

To explore the dedifferentiation phenomenon of human mature adipocytes cultured in vitro and to discuss the possibility of using dedifferentiation adipocytes (DA) as seed cells. Mature adipocytes and ASCs were harvested from human fat aspirates. Mature adipocytes were cultured and induced to DA by ceiling adherent culture method. Cell morphology were observed during the whole process. Viabilities of DA and ASCs were compared by MTT chromatometry and cell growth curves were drawn based on it. Cell surface markers of DA and ASCs were detected by flow cytometry. The adipogenic, osteogenic and chondrogenic ability of DA and ASCs were assessed by oil red O staining, alizarin bordeaux staining and alcian blue staining, respectively. Human mature adipocytes can dedifferentiate into fibroblast-shaped DA. MTT chromatometry assay demonstrated that DA and ASCs both had strong reproductive activity, with no significant difference between them. Flow cytometry assay demonstrated that both DA and ASCs expressed HLA-ABC, CD29 and CD44, while didn't express CD45, CD34 and CD106. After two weeks of adipogenic differentiation, lipid droplets could be displayed by oil red O staining in both DA and ASCs. After two weeks of osteogenic differentiation, calcium salts mineralization in DA and ASCs could be detected by alizarin bordeaux staining. After two weeks of chondrogenic differentiation, matrix of cartilage cells in DA and ASCs could be detected by alcian blue staining. Mature adipocytes can be dedifferentiated into DA in vitro. DA has strong reproductive activity, as well as osteogenic, chondrogenic ability and strong adipogenic ability. It expresses some of the stem cell-related cell surface proteins and is a promising seed cell for adipose tissue engineering.

PCR-Quality DNA Isolation from Human Bronchial Aspirates and Buccal and Eyelid Swabs by a Simple Procedure Based on Alkaline Lysis

There are only a few systematic reports about DNA extraction from routine diagnostic cytological specimens. An inevitable drawback of such techniques is increased spending of time and funds required for obligatory DNA purification. To implement a simple protocol for human DNA isolation from cytological specimens related to lung cancer, bronchial aspirates together with samples collected by swabbing of the inner cheek and eyelid were used. By combining alkaline and temperature lyses it was possible to isolate DNA solution ready for PCR in less than an hour. Testing the method used for amplification of sex chromatin gene fragments showed that it is highly efficient. The presented protocol preserves high-quality DNA that is suitable for PCR-based assays.