Nonadherent Fraction Research Articles

Amyloid Aggregates Are Localized to the Nonadherent Detached Fraction of Aging Streptococcus mutans Biofilms.

ABSTRACTThe number of bacterial species recognized to utilize purposeful amyloid aggregation within biofilms continues to grow. The oral pathogen Streptococcus mutans produces several amyloidogenic proteins, including adhesins P1 (also known as AgI/II, PAc) and WapA, whose truncation products, namely, AgII and AgA, respectively, represent the amyloidogenic moieties. Amyloids demonstrate common biophysical properties, including recognition by Thioflavin T (ThT) and Congo red (CR) dyes that bind to the cross β-sheet quaternary structure of amyloid aggregates. Previously, we observed amyloid formation to occur only after 60 h or more of S. mutans biofilm growth. Here, we extend those findings to investigate where amyloid is detected within 1- and 5-day-old biofilms, including within tightly adherent compared with those in nonadherent fractions. CR birefringence and ThT uptake demonstrated amyloid within nonadherent material removed from 5-day-old cultures but not within 1-day-old or adherent samples. These experiments were done in conjunction with confocal microscopy and immunofluorescence staining with AgII- and AgA-reactive antibodies, including monoclonal reagents shown to discriminate between monomeric protein and amyloid aggregates. These results also localized amyloid primarily to the nonadherent fraction of biofilms. Lastly, we show that the C-terminal region of P1 loses adhesive function following amyloidogenesis and is no longer able to competitively inhibit binding of S. mutans to its physiologic substrate, salivary agglutinin. Taken together, our results provide new evidence that amyloid aggregation negatively impacts the functional activity of a widely studied S. mutans adhesin and are consistent with a model in which amyloidogenesis of adhesive proteins facilitates the detachment of aging biofilms.IMPORTANCE Streptococcus mutans is a keystone pathogen and causative agent of human dental caries, commonly known as tooth decay, the most prevalent infectious disease in the world. Like many pathogens, S. mutans causes disease in biofilms, which for dental decay begins with bacterial attachment to the salivary pellicle coating the tooth surface. Some strains of S. mutans are also associated with bacterial endocarditis. Amyloid aggregation was initially thought to represent only a consequence of protein mal-folding, but now, many microorganisms are known to produce functional amyloids with biofilm environments. In this study, we learned that amyloid formation diminishes the activity of a known S. mutans adhesin and that amyloid is found within the nonadherent fraction of older biofilms. This finding suggests that the transition from adhesin monomer to amyloid facilitates biofilm detachment. Knowing where and when S. mutans produces amyloid will help in developing therapeutic strategies to control tooth decay and other biofilm-related diseases.

Treatment with DC/AML Fusion Vaccine and CD3xCD123 Bi-Specific T-Cell Engager (CD123-CODV-TCE) for Treatment of Acute Myeloid Leukemia

Treatment with DC/AML Fusion Vaccine and CD3xCD123 Bi-Specific T-Cell Engager (CD123-CODV-TCE) for Treatment of Acute Myeloid Leukemia

Bacillus Calmette–Guérin (BCG) vaccine generates immunoregulatory cells in the cervical lymph nodes in guinea pigs injected intra dermally

This work demonstrates the presence of immune regulatory cells in the cervical lymph nodes draining Bacillus Calmette-Guérin (BCG) vaccinated site on the dorsum of the ear in guinea pigs. It is shown that whole cervical lymph node cells did not proliferate in vitro in the presence of soluble mycobacterial antigens (PPD or leprosin) despite being responsive to whole mycobacteria. Besides, T cells from these lymph nodes separated as a non-adherent fraction on a nylon wool column, proliferated to PPD in the presence of autologous antigen presenting cells. Interestingly, addition of as low as 20% nylon wool adherent cells to these, sharply decreased the proliferation by 83%. Looking into what cells in the adherent fraction suppressed the proliferation, it was found that neither the T cell nor the macrophage enriched cell fractions of this population individually showed suppressive effect, indicating that their co-presence was necessary for the suppression. Since BCG induced granulomas resolve much faster than granulomas induced by other mycobacteria such as Mycobacterium leprae the present experimental findings add to the existing evidence that intradermal BCG vaccination influences subsequent immune responses in the host and may further stress upon its beneficial role seen in Covid-19 patients.

Non-Adherent Bone Marrow-Derived Mesenchymal Stem Cells Ameliorate Clinical Manifestations and Inflammation in an Experimental Model of Ulcerative Colitis in Rats.

Background: Functional and developmental versatility of mesenchymal stem cells (MSCs) have generated great interest in their clinical application. Recently, it has been proposed that the non-adherent population of bone marrow cells can differentiate to MSCs in vitro. The present study aimed to compare the anti-inflammatory potentials of adherent and non-adherent MSCs in an experimental model of ulcerative colitis (UC) in rats.Methods: The present experimental study was conducted at the School of Veterinary Medicine, Urmia University (Urmia, Iran) during March-May 2018. UC was induced using acetic acid in three groups of male Wistar rats, namely the control colitis, adherent MSCs treated, and non-adherent MSCs treated groups. Adherent and non-adherent MSCs were collected, characterized, and proliferated. The isolated cells were injected into the peritoneum of the respective groups of colitis rats. After 10 days, the animals were evaluated for gross and microscopic pathology, production of inflammatory mediators, and stress oxidative profile in the gut tissue. The statistical analysis was performed using SPSS software (version 23.0). P<0.05 was considered statistically significant.Results: The non-adherent MSCs had almost similar therapeutic potency compared to the adherent MSCs (P=0.12). They significantly reduced the level of inflammatory mediators and improved the oxidative stress profile in colonic tissue compared to the control colitis group (P=0.0001).Conclusion: The molecular assays and histopathological assessment revealed that the non-adherent MSCs not only had anti-inflammatory and regulatory potency but also enhanced tissue regeneration in UC rats. Therefore, the non-adherent fraction of bone marrow-derived MSCs could be used as a complementary source of MSCs in stem cell therapies.

Involvement of Signaling Cascades in Granulocytopoiesis Regulation under Conditions of Cytostatic Treatment

Suppression of the production of granulocytic CSF under the effect of 5-fluorouracyl is related to disorders in the NF-κB-, cAMP-dependent signaling pathways and MAPK cascade. These secondary messengers are involved in the regulation of functional activity of nonadherent myelokaryocytes starting from day 10 of the experiment (initial period of the hemopoietic granulocytic stem regeneration after antimetabolite challenge). Granulocytic CSF does not play essential role in the formation of colony-stimulating activity of cells of the adherent and nonadherent fractions of the bone marrow. Only cAMP-dependent pathway is involved in the regulation of the realization of the granulocytic precursor growth potential in response to the challenge.

Development and Preliminary Validation of a Feasible Procedure for Isolating RNA from Fiber-Adherent Bacteria in Human Stool.

BackgroundIntestinal bacterial communities are not homogenous throughout the gastrointestinal tract. Human research on the gut microbiome often neglects intra-intestinal variability by relying on a single measurement from stool samples. One source of complexity is the adherence to undigested, residual fiber. Currently, no procedure exists to extract RNA from distinct bacterial subpopulations in stool samples.Material/MethodsA serial centrifugation procedure was developed in which bacterial RNA could be extracted from distinct stool-fractions – fiber-adherent and non-fiber-adherent bacteria. To test whether the separation procedure yielded distinct bacterial subpopulations, a set of RT-qPCR assays were developed for a fiber-adherent bacterial species, Bifidobacterium adolescentis, then a within-subject repeated-measures study was conducted with 3 human subjects undergoing 4 dietary regimens. At each timepoint, between-fraction differences in gene expression were evaluated.ResultsThe RNA isolation procedure was able to isolate intact RNA in 20 of 24 samples in the fiber-adherent fraction. PurB and sdh were identified as suitable reference genes for B. adolescentis RT-qPCR assays. When subjects were provided a high resistant starch diet, bacterial fractions exhibited different expression of the trp operon (p=0.031).ConclusionsOur study provides human gut microbiome researchers a novel tool for evaluating functional characteristics of bacterial subpopulations in human stool. Moreover, these experiments provide modest support for the existence of a functionally unique fiber-adherent subpopulation of B. adolescentis. Until a more thorough evaluation of the adherent and non-adherent fraction can be performed, researchers should be cautious when generalizing functional data derived solely from unfractionated stool samples.

Induced Pluripotent Stem Cells to Model Blood Diseases

Our group is developing induced pluripotent stem cell (iPSC) models of myeloid malignancies, including MDS, MPN and AML. We are generating iPSCs from the bone marrow or blood of patients, which can be maintained indefinitely as pluripotent cell lines and, upon in vitro differentiation along hematopoietic lineages, exhibit hallmark features of these diseases. By integrating mutational analyses with cell reprogramming we can derive iPSCs capturing dominant clones, subclones and normal cells from the same patient and thus have established a collection of iPSC lines representing distinct disease stages along the spectrum of myeloid transformation: predisposition syndromes/preleukemic cells/clonal hematopoiesis; low risk MDS; high risk MDS; and MDS/AML. In parallel, we are using the CRISPR/Cas9 system to introduce or correct mutations in normal or malignant iPSCs, respectively, in isogenic settings and sequential CRISPR gene editing to model mutational cooperation. We recently reported that iPSC lines derived from patients with AML re-establish upon differentiation a leukemic phenotype characterized by extensive proliferation of immature myeloid cells that serially transplant a lethal leukemia into NSG mice (Kotini et al. Cell Stem Cell 2017). Strikingly, we observed that the AML-iPSC-derived hematopoietic stem/progenitor cells (HSPCs) contain two morphologically and immunophenotypically distinct cell subpopulations: a cell fraction (adherent, A) exhibiting adherent growth and containing immature cells with an HSC immunophenotype (CD34+/CD38-/CD90+/CD45RA-/CD49f+); and a non-adherent fraction (suspension, S) of more differentiated cells. Fate-tracking experiments revealed a hierarchical organization, with the A cells renewing themselves and continuously giving rise to the S cells through symmetric and asymmetric divisions. The NSG engraftment potential was largely contained within the adherent cell fraction. Thus, AML-iPSCs exhibit the hallmarks of a leukemia stem cell (LSC) model, namely phenotypic and functional heterogeneity and hierarchical organization, with the A fraction containing LSCs that serially transplant leukemia and give rise to more differentiated cells (S fraction) without engraftment potential. LSCs are believed to be a prominent source of AML relapse, but their rarity and the unavailability of universal and specific immunophenotypic markers prohibits their prospective isolation and makes the study of their properties challenging. This new iPSC-based AML-LSC model enables us for the first time to prospectively obtain large numbers of genetically clonal human LSCs and perform genome-wide integrative molecular analyses and large-scale screening to identify key molecular mechanisms sustaining the properties of LSCs as potential new therapeutic targets. Using this model we characterized the effects of previously proposed compounds with LSC selectivity in self-renewal vs differentiation of LSCs. We also screened a small molecule library of 1280 compounds in the A and S cells in parallel to identify compounds with selectivity for the former as candidates for LSC-specific targeting. DisclosuresNo relevant conflicts of interest to declare.

Simultaneous in vitro generation of human CD34+-derived dendritic cells and mast cells from non-mobilized peripheral blood mononuclear cells

Dendritic cells (DCs) and mast cells (MCs) are key players of the immune system, often coming in close proximity in peripheral tissues. The interplay of these cells is, however, still poorly understood, especially with regards to human cells. The reason for that is the absence of a well established in vitro human cell-based study system that would allow a simultaneous preparation of both cell types. In this study, we show a method for simultaneous generation of DCs and MCs from CD34+ stem cell progenitors that were isolated from the non-adherent fraction of non-mobilized peripheral blood mononuclear cells of healthy donors. We observed that combining stem cells factor (SCF), IL-3 and GM-CSF in serum-free StemPro-34 medium allowed CD34+ cells isolated from an equivalent of 450 ml of peripheral blood to expand to 10–92 × 106 cells after 7 weeks of culturing. These cultures comprised of 6–53% of DCs and 1–21% of MCs as determined by the expression of, respectively, CD11c/HLA-DR or CD117/FcεRI. The DCs were CD1a−CD14−, did not express co-stimulatory molecules CD80 and CD83 and chemokine receptor CCR7. However, the DCs expressed co-stimulatory molecule CD86, and had a capacity to uptake dextran, phagocyte latex particles and induce alloreactivity. MCs, on the other hand, degranulated after crosslinking of FcεRI-bound IgE as determined by the externalization of CD107b. Collectively, our data show that CD34+-derived human DCs and MCs can be generated in a single culture using CD34+ cells isolated from non-mobilized human peripheral blood and that this method may allow ex vivo studies on DC-MC interplay in human system.

Synergistic Integration of Mesenchymal Stem Cells and Hydrostatic Pressure in the Expansion and Maintenance of Human Hematopoietic/Progenitor Cells.

Ex vivo expansion of hematopoietic stem/progenitor cell (HSPC) has been investigated to improve the clinical outcome of HSPC transplantation. However, ex vivo expansion of HSPCs still faces a major obstacle in that HPSCs tend to differentiate when proliferating. Here, we cocultured HSPCs with mesenchymal stem cells (MSCs) and divided the HSPCs into two fractions according to whether they came into adherent to MSCs or not. Additionally, we used hydrostatic pressure (HP) to mimic the physical conditions in vivo. Even nonadherent cells expanded to yield a significantly larger number of total nucleated cells (TNCs), adherent cells maintained the HSPC phenotype (CD34+, CD34+CD38−, and CD133+CD38−) to a greater extent than nonadherent cells and had superior clonogenic potential. Moreover, applying HP significantly increased the number of TNCs, the frequency of the immature HSPC phenotype, and the clonogenic potential. Furthermore, the genetic markers for the HSPC niche were significantly increased under HP. Our data suggest that the nonadherent fraction is the predominant site of HSPC expansion, whereas the adherent fraction seems to mimic the HSPC niche for immature cells. Moreover, HP has a synergistic effect on expansion and functional maintenance. This first study utilizing HP has a potential of designing clinically applicable expansion systems.

Personalized ex vivo multiple peptide enrichment and detection of T cells reactive to multiple tumor-associated antigens in prostate cancer patients

Personalized peptide vaccination is a promising immunotherapeutic approach in prostate cancer (PCa). We therefore examined whether an approach, utilizing personalized multiple peptide-mediated ex vivo enrichment with effector T cells reactive to multiple tumor-associated antigens (TAAs), could be employed as a basis for the development of T cell immunotherapy of PCa. In this study, we used the non-adherent fraction (lymphocytes) of cryopreserved peripheral blood mononuclear cells from a leukapheretic product of biochemically recurrent (BR, n=14) and metastatic hormone-refractory (HR, n=12) PCa patients. The lymphocytes were primed with a pool of mixed overlapping peptides derived from 6 PCa TAAs-PSA, PAP, NY-ESO-1, MAGE-A1, MAGE-A3 and MAGE-A4. After 2weeks of culture, the cells were stimulated with the peptides and T cell reactivity determined by externalization of CD107a. No TAAs-reactive effector T cells were detected in the patient's lymphocytes after their reconstitution. However, following their priming with the TAAs-derived peptides and 2-week culturing, the lymphocytes became enriched with polyclonal TAAs-reactive effector CD8+ T cells in 8 out of 14 BR and 5 out of 12 HR patients. No such reactive CD8+ T cells were detected in cultured lymphocytes without the peptide priming. Stimulation of the responding cultures with peptides derived from individual TAAs revealed a unique repertoire of the reactive CD8+ T cells. Our strategy revealed that the personalized multiple peptide-mediated ex vivo enrichment with multiple TAAs-reactive T cells in the PCa patient's lymphocytes is a viable approach for development of T cell immunotherapy of PCa.

Differential expression of cell cycle and WNT pathway-related genes accounts for differences in the growth and differentiation potential of Wharton\u2019s jelly and bone marrow-derived mesenchymal stem cells

BackgroundIn view of the current interest in exploring the clinical use of mesenchymal stem cells (MSCs) from different sources, we performed a side-by-side comparison of the biological properties of MSCs isolated from the Wharton’s jelly (WJ), the most abundant MSC source in umbilical cord, with bone marrow (BM)-MSCs, the most extensively studied MSC population.MethodsMSCs were isolated and expanded from BM aspirates of hematologically healthy donors (n = 18) and from the WJ of full-term neonates (n = 18). We evaluated, in parallel experiments, the MSC immunophenotypic, survival and senescence characteristics as well as their proliferative potential and cell cycle distribution. We also assessed the expression of genes associated with the WNT- and cell cycle-signaling pathway and we performed karyotypic analysis through passages to evaluate the MSC genomic stability. The hematopoiesis-supporting capacity of MSCs from both sources was investigated by evaluating the clonogenic cells in the non-adherent fraction of MSC co-cultures with BM or umbilical cord blood-derived CD34+ cells and by measuring the hematopoietic cytokines levels in MSC culture supernatants. Finally, we evaluated the ability of MSCs to differentiate into adipocytes and osteocytes and the effect of the WNT-associated molecules WISP-1 and sFRP4 on the differentiation potential of WJ-MSCs.ResultsBoth ex vivo-expanded MSC populations showed similar morphologic, immunophenotypic, survival and senescence characteristics and acquired genomic alterations at low frequency during passages. WJ-MSCs exhibited higher proliferative potential, possibly due to upregulation of genes that stimulate cell proliferation along with downregulation of genes related to cell cycle inhibition. WJ-MSCs displayed inferior lineage priming and differentiation capacity toward osteocytes and adipocytes, compared to BM-MSCs. This finding was associated with differential expression of molecules related to WNT signaling, including WISP1 and sFRP4, the respective role of which in the differentiation potential of WJ-MSCs was specifically investigated. Interestingly, treatment of WJ-MSCs with recombinant human WISP1 or sFRP4 resulted in induction of osteogenesis and adipogenesis, respectively. WJ-MSCs exhibited inferior hematopoiesis-supporting potential probably due to reduced production of stromal cell-Derived Factor-1α, compared to BM-MSCs.ConclusionsOverall, these data are anticipated to contribute to the better characterization of WJ-MSCs and BM-MSCs for potential clinical applications.

The expression of Helicobacter pylori tfs plasticity zone cluster is regulated by pH and adherence, and its composition is associated with differential gastric IL-8 secretion.

Helicobacter pylori virulence is associated with different clinical outcomes. The existence of an intact dupA gene from tfs4b cluster has been suggested as a predictor for duodenal ulcer development. However, the role of tfs plasticity zone clusters in the development of ulcers remains unclear. We studied several H. pylori strains to characterize the gene arrangement of tfs3 and tfs4 clusters and their impact in the inflammatory response by infected gastric cells. The genome of 14 H. pylori strains isolated from Western patients, pediatric (n=10) and adult (n=4), was fully sequenced using the Illumina platform MiSeq, in addition to eight pediatric strains previously sequenced. These strains were used to infect human gastric cells, and the secreted interleukin-8 (IL-8) was quantified by ELISA. The expression of virB2, dupA, virB8, virB10, and virB6 was assessed by quantitative PCR in adherent and nonadherent fractions of H. pylori during in vitro co-infection, at different pH values. We have found that cagA-positive H. pylori strains harboring a complete tfs plasticity zone cluster significantly induce increased production of IL-8 from gastric cells. We have also found that the region spanning from virB2 to virB10 genes constitutes an operon, whose expression is increased in the adherent fraction of bacteria during infection, as well as in both adherent and nonadherent fractions at acidic conditions. A complete tfs plasticity zone cluster is a virulence factor that may be important for the colonization of H. pylori and to the development of severe outcomes of the infection with cagA-positive strains.

Adherence to ASTRO ‘Choosing Wisely’ recommendations for radiation therapy in low-risk endometrial cancer.

240 Background: The American Society for Radiation Oncology (ASTRO) ‘Choosing Wisely’ (CW) list notes radiation interventions lacking evidence of benefit in cancer care. This study characterizes adherence to the 2014 CW item to ‘not recommend radiation following hysterectomy in low-risk endometrial cancer’ (Stage I, Grade 1-2). No studies have previously evaluated adherence to this CW item. Methods: SEER records for patients age &gt;65 at first primary diagnosis of Stage I (Grade 1-2) endometrial cancer between 2008-2011 were linked with Medicare files. Included cases had Medicare Part A and B coverage for ≥12 months before/after diagnosis and had a hysterectomy 1 month prior to 4 months following diagnosis. We used logistic regression to evaluate associations between socio-demographic and clinical characteristics (Table 1) and receipt of radiation therapy within 1 year of diagnosis. Results: Among 2,938 women meeting eligibility criteria, mean age was 74, 90% were white, and 55% lived in a ‘big metro’ area (population &gt;1m). The non-adherent fraction was 24%. Among non-adherent cases, 76% received brachytherapy. In adjusted analyses (Table 1), only tumor grade was associated with non-adherence. Conclusions: More than 20% of low-risk endometrial cancer cases in our sample were non-adherent to ASTRO CW recommendations, and this only varied significantly by tumor grade. Therefore, use of radiation therapy following hysterectomy appears to be a widespread issue. This is important because non-adherence may result in side effects or substantial cost without commensurate clinical benefit. This study’s follow-up period was prior to the ASTRO CW list, but similar recommendations existed in ASCO, ESMO and NCCN guidelines at the time—suggesting work is need to align practice with guidelines. Our findings provide a baseline measure against which future studies can measure progress. [Table: see text]

Magnetic stromal layers for enhanced and unbiased recovery of co-cultured hematopoietic cells

Cell co-culture systems have a long history of application in hematology and hold promise for successful hematopoietic stem and progenitor cell expansion. Here we report that various types of stromal cells used in such co-cultures can be rapidly and efficiently labeled with l-lysine-modified Fe3O4 magnetic nanoparticles. Hematopoiesis-supporting activity does not seem to be compromised after magnetic labeling of stromal cells, and the loss of the label by stromal layers during extended culturing is negligible. Magnetic labeling allows for simple and efficient removal of stromal component, yielding unbiased hematopoietic cell populations. When Lin– bone mouse marrow fraction was co-cultured with magnetic stromal layers and resulting cell populations were harvested by trypsinization, the yields of total nucleated cells, colony forming cells, and phenotypically primitive Lin–Sca-1+c-kit+ subset were substantially higher as compared with nonadherent cell fractions harvested after conventional stromal co-culture. The advantage offered by the magnetic stroma approach over the traditional one was even more significant after a second round of co-culture and was more dramatic for more primitive hematopoietic cells. We conclude that magnetic stromal layers represent a simple, efficient, and convenient tool for co-culturing and subsequent recovery of sufficiently pure unbiased populations of hematopoietic cells.

Injured hepatocyte-released microvesicles induce bone marrow-derived mononuclear cells differentiation

The ability of bone marrow-derived mononuclear cells (BMMCs) to differentiate into hepatocyte-like cells under different conditions has been demonstrated previously. In the present study, we investigated the effect of CCl4-injured hepatocytes on the differentiation of the non-adherent (NAD) fraction of BMMCs. Differentiation (cell fate) was analyzed after 2, 6 and 24h of co-culture by gene and protein expression and by urea production. We also evaluated the presence of microvesicles (MVs) in the supernatant of differentiated cells, their content and the ability of these cells to absorb them. Hepatocyte-like characteristics were observed in the NAD cells after 24h of co-culture with injured hepatocytes. Cells that were co-cultured with healthy hepatocytes did not present signs of differentiation at any analyzed time point. Analysis of the supernatant from differentiated cells revealed the presence of MVs carrying hepatocyte-specific mRNAs, including Albumin, Coagulation factor V, Alpha-fetoprotein, and Cytokeratin 18. The incorporation of injured hepatocyte-derived MVs by NAD cells was shown at 24h, suggesting a possible role for MVs in the induction of cell plasticity.

Suspension-Expansion of Bone Marrow Results in Small Mesenchymal Stem Cells Exhibiting Increased Transpulmonary Passage Following Intravenous Administration.

Mesenchymal stem cells (MSCs) have been extensively explored in a variety of regenerative medicine applications. The relatively large size of MSCs expanded in tissue culture flasks leads to retention in the microcirculation of the lungs following intravenous delivery, reducing their capacity to reach target sites. We explored whether the expansion of whole marrow in suspension cultures would yield smaller MSCs with increased capacity to traverse the pulmonary microcirculation compared with traditional monolayer cultures. We tested this hypothesis using rat marrow in a suspension bioreactor culture with fibronectin-coated microcarriers, leading to sustained expansion of both the microbead-adherent cells, as well as of a nonadherent cell fraction. Magnetic depletion of CD45(+) cells from the bioreactor cultures after 5 weeks led to a highly enriched CD73(+)/CD90(+)/CD105(+) MSC population. The bioreactor-grown MSCs were significantly smaller than parallel monolayer MSCs (15.1 ± 0.9 μm vs. 18.5 ± 2.3 μm diameter, p<0.05). When fluorescently labeled bioreactor-grown MSCs were intravenously injected into rats, the peak cell concentration in the arterial circulation was an order of magnitude higher than similarly delivered monolayer-grown MSCs (94.8 ± 29.6 vs. 8.2 ± 5.6/10(6) nucleated blood cells, respectively, p<0.05). At 24 h after intravenous injection of the LacZ-labeled bioreactor-grown MSCs, there was a significant threefold decrease in the LacZ-labeled MSCs trapped in the lungs, with a significant increase in the cells reaching the spleen and liver in comparison to their monolayer MSC counterparts. Bioreactor-grown whole marrow cell cultures yielded smaller MSCs with increased capacity to traverse the pulmonary microcirculation compared with traditionally expanded monolayer MSCs. This may significantly improve the capacity and efficiency of these cells to home to injury sites downstream of the lungs.

CD25 (IL-2R) expression correlates with the target cell induced cytotoxic activity and cytokine secretion in human natural killer cells.

Cytotoxic activity is one of the major functions of Natural Killer (NK) cells and is a critical effector mechanism of innate immune responses against infected or cancer cells. A variety of assays have been developed to determine NK cell cytotoxic activity, however a receptor-based screening tool is still lacking. Here, we propose the CD25 receptor as a candidate for NK cell cytotoxicity marker. We have verified that there is a correlation between classic target cell induced cytotoxicity markers and the CD25 expression on NK cells. Non-adherent lymphocyte fractions pre-stimulated with Escherichia coli O55:B5 lipopolysaccharide were co-cultured with settled HeLa targets in a four hour long cytotoxic assay. The cytotoxic effect was evaluated by MTT reduction assay and quantification of soluble cytotoxicity markers (granzyme B, FasL, caspase-8, IFN-γ and IL-2) was done by ELISA. Lymphocytes were stained with anti-CD3-Cy-5, anti-CD56/CD16/Nkp46-FITC and anti-CD25-PE antibodies and analyzed by flow cytometry. We observed that the CD25 expression exclusively on the CD3(-)CD56(+)CD25(+) NK cells was positively correlated with their cytotoxic function evaluated by the MTT test (r = 0.68), the upregulation of granzyme B (r = 0.89), IL-2 (r = 0.78) and IFN-γ (r = 0.57), however, it was not positively correlated with FasL and caspase-8. We conclude that the CD25 expression might serve as an in vitro receptor-based screening tool for NK cell activity.

Hematopoietic Origin of Murine Lung Fibroblasts.

Multiple origins, including the bone marrow, have been suggested to contribute to fibroblast populations in the lung. Using bone marrow reconstitution strategies, the present study tested the hypothesis that the bone marrow hematopoietic stem cell (HSC) gives rise to lung tissue fibroblasts in vivo. Data demonstrate that the nonadherent bone marrow fraction is enriched for CD45+ HSC-derived cells and was able to reconstitute hematopoiesis in lethally irradiated animals. Analysis of peripheral blood and lung tissues from engrafted mice demonstrated the ability of this population to give rise to CD45+/Discoidin-Domain Receptor-2+ (DDR2) circulating fibroblast precursors (CFPs) in blood and fibroblast populations in lung. An HSC origin for lung fibroblasts was confirmed using a novel clonal cell transplantation method in which the bone marrow is reconstituted by a clonal population derived from a single HSC. Together, these findings provide evidence for an HSC contribution to lung fibroblasts and demonstrate a circulating intermediate through the CD45+/DDR2+ HSC-derived CFP.

Application of chimeric glucanase comprising mutanase and dextranase for prevention of dental biofilm formation.

Water-insoluble glucan (WIG) produced by mutans streptococci, an important cariogenic pathogen, plays an important role in the formation of dental biofilm and adhesion of biofilm to tooth surfaces. Glucanohydrolases, such as mutanase (α-1,3-glucanase) and dextranase (α-1,6-glucanase), are able to hydrolyze WIG. The purposes of this study were to construct bi-functional chimeric glucanase, composed of mutanase and dextranase, and to examine the effects of this chimeric glucanase on the formation and decomposition of biofilm. The mutanase gene from Paenibacillus humicus NA1123 and the dextranase gene from Streptococcus mutans ATCC 25175 were cloned and ligated into a pE-SUMOstar Amp plasmid vector. The resultant his-tagged fusion chimeric glucanase was expressed in Escherichia coli BL21 (DE3) and partially purified. The effects of chimeric glucanase on the formation and decomposition of biofilm formed on a glass surface by Streptococcus sobrinus 6715 glucosyltransferases were then examined. This biofilm was fractionated into firmly adherent, loosely adherent, and non-adherent WIG fractions. Amounts of WIG in each fraction were determined by a phenol-sulfuric acid method, and reducing sugars were quantified by the Somogyi-Nelson method. Chimeric glucanase reduced the formation of the total amount of WIG in a dose-dependent manner, and significant reductions of WIG in the adherent fraction were observed. Moreover, the chimeric glucanase was able to decompose biofilm, being 4.1 times more effective at glucan inhibition of biofilm formation than a mixture of dextranase and mutanase. These results suggest that the chimeric glucanase is useful for prevention of dental biofilm formation.

Pacritinib Suppresses Leukemic Outgrowth from FLT3-ITD Positive Stroma-Adherent Primary AML Cells

Background: Targeting the stroma-AML cell interaction is a new dimension in anti-leukemia therapy. FLT3-ITD AML cells are known to derive protection from bone marrow stroma through up-regulation of resistance pathways such as MEK and JAK2. Pacritinib is a dual FLT3 and JAK2 inhibitor with equipotent activity for each target and is currently offered as stand-alone therapy for relapsed AML patients with FLT3-ITD mutant disease enrolled in the MRC AML 17 trial.Aim: We set up this study to determine if pacritinib as a single agent is sufficient to abrogate the stroma mediated resistance of AML, and to investigate leukemic signalling in stroma-adherent AML cells.Results: Pacritinib efficacy was assessed in 62 primary AML samples. FLT3-ITD samples were significantly more sensitive to pacritinib compared to FLT3 wild type AML samples after 48 hours exposure in vitro (mean IC50 92 nM vs 292 nM [range ITD 11-290 nM, range WT 19-2600 nm], p=0.01). Pacritinib induced apoptosis in AML cells but was not lethal to normal CD34+ bone marrow cells within the same dose range. FLT3-ITD AML cells cultured on MS5 stroma retained sensitivity to pacritinib after 7 days of co-culture (p<0.01, n=6).There was no significant effect on stromal cells. In this study, an MS5 stromal co-culture model was set up to enable interrogation of FLT3-ITD mutated primary AML cells that are adherent and non-adherent to stroma. PCR analyses performed on CD45+ adherent AML cells confirmed that they carried FLT3-ITD. Flow analyses demonstrated that the reduction of stroma-adherent AML cells was pacritinib dose dependent. Following 7 days of treatment and after drug wash-out, prior pacritinib exposure resulted in sustained suppression of leukemic outgrowth from stroma-adherent FLT3-ITD AML cells at 14 days of co-culture, compared to untreated cells (p<0.01, n=6). To establish if pacritinib could impact upon longer-term leukemic stromal adherence and survival in FLT3-ITD AML, we stained leukemic cells with Aldefluor and flow sorted the CD34+ 38- ALDH intermediate fraction, which has been shown to possess in vivo engrafting ability and to correlate with clinical relapse. Sorted cells were plated onto MS5 stroma and cobblestone area forming cell (CAFC) capacity determined as a function of pacritinib dose. Pacritinib reduced CAFC frequency at 5 weeks (from 500 nm, n=3).To investigate leukemic cell signalling, FLT3-ITD mutant AML cells were cultured on stroma in the presence of pacritinib for up to 24 hours, and intracellular flow analyses performed on adherent and non-adherent fractions. Control stroma-free cultures demonstrated significant p-STAT5 knockdown from 1 hour of drug exposure. In the presence of stroma, non-adherent AML cells did not show this reduction in p-STAT5. P-STAT5 down-regulation in adherent AML cells was delayed (to 18 hours), but exhibited dose dependency (p<0.05 at 500 nm pacritinib, p=0.0005 at 1 μM; n=6). Interrogation of JAK2 signalling showed up-regulation of p-JAK2 in adherent AML cells in the presence of pacritinib from 3 hours in 2 of 5 samples. There was no abrogation of AKT or ERK signalling in stroma-adherent or non-adherent fractions and this was confirmed by immuno-blotting. Interestingly, pacritinib was observed to decrease non-phosphorylated beta catenin levels in stroma-adherent AML cells and this was not seen in non-adherent AML cells (p=0.029, n=5).Short-term drug response Cell Glo luminescent assays reflected results from intracellular signalling experiments. Significant synergy was seen when pacritinib was combined with a MEK inhibitor (PD0325901) with accompanying down-regulation of ERK (mean CI 0.31 [range 0.19-0.44] at 5:1 ratio, n=20).Conclusion: These results demonstrate that pacritinib can suppress leukemic outgrowth from stroma adherent cells in FLT3-ITD driven AML in medium-term (14 days) and long-term (5 weeks) assays. However, short-term intracellular signalling profiles suggest that resistance pathways can remain active in some cases and may require AKT/ERK targeting for their eradication. Good synergy is seen with MEK inhibition, making this an attractive combination for clinical evaluation. DisclosuresNo relevant conflicts of interest to declare.