Growth Of Colony-forming Units Research Articles

Abstract PO4-28-01: Molecular profiling revealed activation of cytokine signaling and suppression of lipid metabolism as the hallmarks of doxorubicin-resistant TNBC

Abstract Background: Chemotherapeutic resistance observed in triple-negative breast cancer (TNBC) poses a substantial clinical hurdle, emphasizing the imperative to enhance our comprehension of the underlying mechanisms with the aim of potentially mitigating or reversing this phenomenon. Methods: Doxorubicin-resistant (DoxR) TNBC models (MDA-MB-231 and BT-549) were established through continuous exposure to increasing concentrations of doxorubicin. RNA-Seq was conducted using the Illumina platform, while bioinformatics analyses were performed using CLC genomics workbench 20.2 and iDEP.91. Colony forming unit (CFU), flow cytometry and fluorescent microscopy were used to assess cell proliferation, cell cycle distribution, and cell death, respectively. Western blotting was used to confirm the expression and phosphorylation of protein targets. Ingenuity pathway analysis (IPA) and STRING database v 11.5 were used for network and pathway analyses, while Kaplan-Meier Plotter was used for survival analyses. Results: Herein, we provide a comprehensive functional and transcriptomic characterization of DoxR TNBC models, revealing multiple affected signaling networks and functional categories. The defense and immune response, response to stress, response to cytokine and external stimulus and cytokine-mediated signaling pathways were among the top activated, while suppression of cholesterol, sterol and lipid biosynthesis and metabolism were the hallmarks of DoxR cells. Upstream regulator analysis revealed IL-1B cytokine network activation in DoxR cells. Concordantly, disease and function analysis on the downregulated genes in DoxR TNBC cells revealed the most pronounced enrichment in pathways associated with cell invasion, colony formation, as well as lipid and carbohydrate metabolism. A number of upregulated genes were validated by qRT-PCR, which correlated with unfavorable overall survival (OS). Functionally, DoxR cells exhibited remarkable suppression of cell proliferation, as evidenced by diminished CFU potential, sphere formation and growth under 3D organoid culture, and cell migration. Although DoxR TNBC cells exhibited a slow cell cycling under normal culture conditions, wild-type TNBC cells exhibited remarkable arrest in the G2M phase when exposed to doxorubicin, which was less evident in DoxR TNBC models, suggesting DoxR cells undergo a quiescent state in response to chemotherapeutic challenge. In agreement with those data, increased phosphorylation of CHK2 and p53 proteins was remarkable in DoxR cells, thus promoting cellular dormancy. Conclusion: Our data revealed cellular dormancy and suppression of lipid metabolism as the hallmark associated with doxorubicin-resistant TNBC cells. On the other hand, cytokine signaling and stress response were the most enriched functional categories in DoxR TNBC cells. Therapeutic targeting to reverse the quiescent state or suppression of cytokine signaling networks could enhance the efficacy of doxorubicin-based therapeutics. Citation Format: Vishnubalaji Radhakrishnan, Nehad Alajez. Molecular profiling revealed activation of cytokine signaling and suppression of lipid metabolism as the hallmarks of doxorubicin-resistant TNBC [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-01.

Abstract 11 Monitoring Cord Blood Inventory Reliability by Key Quality Indicators At and After Release

Abstract Introduction As per FACT standards, Cord Blood Banks (CBB) have to verify that they provide a safe and effective product for transplant. Validated processing methods and storage conditions are the first steps towards this goal, but what are the final steps to monitor the quality of the cord blood units (CBU) released for transplant? Objectives We present here a retrospective analysis of our Program’s final verification process to meet this FACT requirement. Methods CBU shipped between January 2018 and December 2022 were reviewed for out of specification events. The following Quality Control (QC) parameters were measured on attached segments before shipment: total nucleated cells (TNC), viable CD34 cells (vCD34), and colony forming units (CFU). Confirmed date of infusion and engraftment data were obtained from CIBMTR. Results A total of 619 CBU were shipped during the study period. 550 had a segment analyzed at the time of shipment. Average post-thaw CD34 viability on the segments was 93.5% (Table 1). Average recoveries for TNC, viable CD34 and CFU were 83.3%, 64.90% and 60.86%, respectively (Table 1). 6 (1%) CBUs were found to have CD34 viability lower than FACT requirement (≥70%) (Table 1). Investigations were performed for each CBU, concluding that these were technical issues with no repercussion on the final product. No segments demonstrated absence of CFU growth. 544 CBUs had an infusion date recorded by CIBMTR (Table 1). 250 of the CBU infused had engraftment data, and 242 engrafted. 8 (3%) CBU transplants resulted in non-engraftment. All had QC results within expected range (Table 1). Discussion Our QC results confirm the continued stability of the CBU until shipment. The small subset of CBU with out of specification results were linked to technical issues and did not affect the outcome of transplant. Outcome data is another way of assessing the quality of the CBUs, even though it may be influenced by many factors out of the CBB’s responsibility (such as diagnosis, conditioning, multiple transplants, etc…). The combined analysis of the two datasets is a good way for a CBB to ensure processes are performing as expected over time and monitor the quality of the final product.

MP05-12 IN VITRO EFFECT OF E.COLI PHAGES ON E.COLI BIOFILM MODEL BUILT ON URETERAL STENT

You have accessJournal of UrologyCME1 Apr 2023MP05-12 IN VITRO EFFECT OF E.COLI PHAGES ON E.COLI BIOFILM MODEL BUILT ON URETERAL STENT Leonid Boyarsky, Ortal Yerushalmi, Ronen Hazan, Amitay Lorber, Vladimir Yutkin, Ofer Gofrit, and Mordechai Duvdevani Leonid BoyarskyLeonid Boyarsky More articles by this author , Ortal YerushalmiOrtal Yerushalmi More articles by this author , Ronen HazanRonen Hazan More articles by this author , Amitay LorberAmitay Lorber More articles by this author , Vladimir YutkinVladimir Yutkin More articles by this author , Ofer GofritOfer Gofrit More articles by this author , and Mordechai DuvdevaniMordechai Duvdevani More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003216.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: To assess, in vitro, the ability of E.coli phages to eradicate biofilms built on ureteral stents with an intention to develop an additional treatment option for chronic urinary infection in patients with indwelling ureteral stent. METHODS: Phages were grown on isolated E.coli cultures. Phage host range of infection was determined with plaque assay method. The urine stability of phages was assessed. Biofilm model was built separately on 96 well plates and on ureteral stent. Following biofilm infection, the phage lytic with and without ampicillin was assessed by colony forming units (CFU) test and scanning electron microscopy (SEM). RESULTS: Ampicillin resistant, E.coli strain O5 was found as sensitive to a group of phages designated ECOY 1-8 and was therefore selected as a study strain. ECOY5 was found to be stable in urine and was selected as a study phage. In the study group, the CFU test showed 10- fold and 100- fold decrease in E.coli CFU growth for well plate and stent models respectively. The lytic effect of phage treatment was visualized on SEM images. CONCLUSIONS: Use of the ECYO5 phage alone or in combination with ampicillin appears to be very effective in breaking down the biofilm of E.coli. Further research is needed to develop phage-based products for stent associated infections including other uropathogens. Source of Funding: none © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e48 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Leonid Boyarsky More articles by this author Ortal Yerushalmi More articles by this author Ronen Hazan More articles by this author Amitay Lorber More articles by this author Vladimir Yutkin More articles by this author Ofer Gofrit More articles by this author Mordechai Duvdevani More articles by this author Expand All Advertisement PDF downloadLoading ...

Toxicity of nZVI in the growth of bacteria present in contaminated soil.

The use of nano zero-valent iron (nZVI) for the remediation of degraded areas is a consolidated practice. However, the long-term reactions that occur in the environment remain unknown. This study aimed to evaluate the potential toxic effects on the growth of colony-forming units (CFUs) of Bacillus cereus and Pseudomona aeruginosa present in soil contaminated with hexavalent chromium (Cr6+) and pentachlorophenol (PCP) nanoremediated with nZVI. The treatments were natural soil (control), soil contaminated by Cr6+, soil contaminated by PCP, and soil contaminated by Cr6+ and PCP (Cr6+ and PCP), all in duplicate. The concentration of contaminants used was 100mg/kg of soil. One of the drums of the duplicate received an injection of nZVI solution with a concentration of 50g/kg. Analysis was performed 7, 15, 21, 30, 60, and 90 days after the nZVI injection. Temporary oscillations in the abundance of the microbiological community were observed, characterizing the adaptation of bacteria to the contaminants. The bacteria showed similar behavior. Ninety days after the injection of nZVI, the averages of the CFUs were statistically equal, with the lowest coefficient of variation and the highest concentration of CFUs occurring. The strains of B. cereus and P. aeruginosa were resistant to the concentrations of nZVI, Cr6+, and PCP. The nanoremediation of nZVI in soil contaminated by Cr6+ and PCP had no toxic effects on the population of the bacteria evaluated and did not present major disturbances in temperature, electrical conductivity, pH, and humidity over time.

Influence of Staphylococcus epidermidis biofilm on the mechanical strength of soft tissue allograft.

We sought to determine the impact of bacterial inoculation and length of exposure on the mechanical integrity of soft tissue tendon grafts. Cultures of Staphylococcusepidermidis were inoculated on human tibialis posteriorcadaveric tendon to grow biofilms. A low inoculum in 10% growth medium was incubated for 30 min to replicate conditions of clinical infection. Growth conditions assessed included inoculum concentrations of 100, 1000, 10,000 colony-forming units(CFUs). Tests using the MTS Bionix system were performed to assess the influence of bacterial biofilms on tendon strength. Load-to-failure testing was performed on the tendons, and the ultimate tensile strength was obtained from the maximal force and the cross-sectional area. Displacements of tendon origin to maximal displacement were normalized to tendon length to obtain strain values. Tendon force-displacement and stress-strain relationships were calculated, and Young's modulus was determined. Elastic modulus and ultimate tensile strength decreased with increasing bioburden. Young's modulus was greater in uninoculated controls compared to tendons inoculated at 10,000 CFU (p = 0.0011) but unaffected by bacterial concentrations of 100 and 1000 CFU (p = 0.054, p = 0.078). Increasing bioburden was associated with decreased peak load to failure (p = 0.043) but was most significant compared to the control under the 10,000and 1000 CFU growth conditions (p = 0.0005, p = 0.049). The presence of S. epidermidis increased elasticity and decreased ultimate tensile stress of human cadaveric tendons, with increasing effect noted with increasing bioburden.

Ex vivo expansion and functional activity preservation of adult hematopoietic stem cells by a diarylheptanoid from Curcuma comosa

Hematopoietic stem cells (HSCs, CD34+ cells) have shown therapeutic efficacy for transplantation in various hematological disorders. However, a large quantity of HSCs is required for transplantation. Therefore, strategies to increase HSC numbers and preserve HSC functions through ex vivo culture are critically required. Here, we report that expansion medium supplemented with ASPP 049, a diarylheptanoid isolated from Curcuma comosa, and a cocktail of cytokines markedly increased numbers of adult CD34+ cells. Interestingly, phenotypically defined primitive HSCs (CD34+CD38-CD90+) were significantly increased under ASPP 049 treatment relative to control. ASPP 049 treatment also improved two functional properties of HSCs, as evidenced by an increased number of CD34+CD38- cells in secondary culture (self-renewal) and the growth of colony-forming units as assessed by colony formation assay (multilineage differentiation). Transplantation of cultured CD34+ cells into immunodeficient mice demonstrated the long-term reconstitution and differentiation ability of ASPP 049-expanded cells. RNA sequencing and KEGG analysis revealed that Hippo signaling was the most likely pathway involved in the effects of ASPP 049. These results suggest that ASPP 049 improved ex vivo expansion and functional preservation of expanded HSCs. Our findings provide a rationale for the use of ASPP 049 to grow HSCs prior to hematological disease treatment.

Final Endodontic Irrigation with 2% Peracetic Acid: Antimicrobial Activity and Cytotoxicity.

Objective The aim of present study was to assess the cytotoxicity and antimicrobial efficacy of 2% peracetic acid (PAA) compared with 5.25% sodium hypochlorite (NaOCl) and 2% chlorhexidine (CHX). Material and Methods For the cytotoxicity test, 100 µl of the tested solutions were added in 12 wells with ECV 304 endothelial cells in each group: NaOCl, CHX, and PAA, in addition to the control group. Each solution was evaluated after 24 hours of contact in four dilutions: 0.2, 0.1, 0.05 and 0.025 through mitochondrial function using MTT colorimetric assay. In the antimicrobial evaluation, 40 dentin blocks 5 mm in length and 0.2 g in weight were incubated with 400 µl of Enterococcus faecalis suspension for 21 days at 37°C. The contaminated samples were divided into three experimental groups within 5 minutes of contact: NaOCl group, CHX group, PAA group, as well as the positive control group. The specimens received treatment and were transferred to a tube with saline for serial dilution of the solution and seeding for isolation and colony forming unit (CFU) count. Statistical Analysis The results obtained were expressed as mean (A570 nm) ± standard deviation (SD) and in a multiple linear regression model and multiple comparisons conducted. Results The antimicrobial evaluation revealed that the NaOCl and CHX groups showed a statistically significant difference compared with the control group ( p < 0.001), while the PAA reduced only the CFU growth. It can be concluded that, among the agents tested, PAA expressed greater cell viability, followed by CHX and NaOCl. However, it did not show greater antimicrobial activity in vitro in the mature biofilm of Enterococcus faecalis.

Different Antifungal Activity of Anabaena sp., Ecklonia sp., and Jania sp. against Botrytis cinerea.

Water extracts and polysaccharides from Anabaena sp., Ecklonia sp., and Jania sp. were tested for their activity against the fungal plant pathogen Botrytis cinerea. Water extracts at 2.5, 5.0, and 10.0 mg/mL inhibited B. cinerea growth in vitro. Antifungal activity of polysaccharides obtained by N-cetylpyridinium bromide precipitation in water extracts was evaluated in vitro and in vitro at 0.5, 2.0, and 3.5 mg/mL. These concentrations were tested against fungal colony growth, spore germination, colony forming units (CFUs), CFU growth, and on strawberry fruits against B. cinerea infection with pre- and post-harvest application. In in vitro experiments, polysaccharides from Anabaena sp. and from Ecklonia sp. inhibited B. cinerea colony growth, CFUs, and CFU growth, while those extracted from Jania sp. reduced only the pathogen spore germination. In in vitro experiments, all concentrations of polysaccharides from Anabaena sp., Ecklonia sp., and Jania sp. reduced both the strawberry fruits infected area and the pathogen sporulation in the pre-harvest treatment, suggesting that they might be good candidates as preventive products in crop protection.

A novel thrombopoietin mimetic RWJ-800088 increases megakaryopoiesis without causing malignant proliferation in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML).

e18527 Background: In patients (pts) with AML and MDS, thrombocytopenia is a source of morbidity/mortality and there is a need for safer treatment options. RWJ-800088 (RWJ) is a novel pegylated thrombopoietin (TPO) mimetic peptide that increases megakaryopoiesis preclinically and in humans. Before using a TPO mimetic agent in AML and MDS pts, it is important to evaluate if the agent has potential stimulatory effects on malignant myeloid cells. To assess whether RWJ is likely to stimulate malignant myeloid cells, we evaluated its effects in vitro on primary human samples from healthy controls and AML/MDS pts. Methods: We studied the effect of various RWJ doses on proliferation of AML/MDS cell lines; clonogenic capacity for leukemic colony forming units (CFU) and megakaryocytic CFU (CFU-Mk) of mononuclear cells (MNC) from controls and several MDS and AML pts; and expression of differentiation markers in MNC cultures. Results: RWJ treatment had no effect on proliferation of THP1, MOLM13, MV411 and MDSL cell lines at doses up to 1000 ng/ml. A slight increase in proliferation of CMK, a megakaryocytic cell line, was seen at doses &gt; 5xEC50 (mean 12%, p = 0.03). Primary clonogenic assays from 18 AML/MDS pts did not show increased growth of leukemic CFU at RWJ concentrations up to 10 ng/ml and RWJ had a nonsignificant inhibitory effect in 4 pts. Notably, RWJ significantly increased normal CFU-Mk formation from MNC in 6 of 8 AML and all MDS pts (mean 2.6-fold, p = 0.02), as well as from healthy CD34+ stem cells and healthy MNC controls (mean 1.7-fold, p = 0.03). FACS analysis of primary cultures treated with RWJ showed no increase in the malignant blast compartment, no change in differentiation markers of granulocyte/monocyte or erythrocyte lineages but a significant increase in the number of CD41a+ megakaryocytic cells (mean 1.8-fold, p &lt; 0.01). RWJ treatment increased phosphorylation of STAT5 but not STAT3, demonstrating on target stimulation of the TPO receptor in hematopoietic cells. Conclusions: Our results show that the novel TPO mimetic RWJ stimulates megakaryopoiesis without causing proliferation of the malignant myeloid clone in MDS and AML.

Prospective assessment of the effectiveness of standard high-level disinfection for echoendoscopes

Duodenoscopes have been implicated in the transmission of multidrug-resistant organisms (MDROs). Echoendoscopes could potentially transmit infection. The aim of this study was to assess the effectiveness of standard high-level disinfection (HLD) for radial and linear echoendoscopes and to compare it with that of duodenoscopes. We performed a prospective single-center study sampling echoendoscopes immediately before use, from the working channel (radial and linear echoendoscopes) and the transducer (radial echoendoscope) or elevator mechanism and transducer (linear echoendoscope). The primary outcome was the proportion of echoendoscopes with any culture showing≥1 MDRO; secondary outcomes included bacterial growth >0 colony forming units (CFUs) and≥10 CFUs on either sampling location. We compared these findings with duodenoscope cultures from the previously published DISINFECTS trial. During the study period, 101 echoendoscopes were sampled (n= 50 radial echoendoscopes, n= 51 linear echoendoscopes). No MDROs were recovered. Bacterial growth >0 CFUs was noted in 6% and≥10 CFUs in 3% of all echoendoscopes. There was no significant difference in growth between radial and linear echoendoscopes (P= .4 for >0 CFU growth; P= .6 for≥10 CFUs growth). The proportion of transducer and/or elevator mechanism positive forbacterial growth was significantly higher in duodenoscopes as compared with echoendoscopes (P= .02). After standard HLD, no echoendoscope showed MDRO growth, 6% showed >0 CFUs, and 3% showed≥10 CFUs bacterial growth. Bacterial growth was higher in duodenoscopes at the level of the transducer and/or elevator mechanism when compared with echoendoscopes.

Is Marine Dispersion of the Lethargic Crab Disease Possible? Assessing the Tolerance of Exophiala cancerae to a Broad Combination of Salinities, Temperatures, and Exposure Times.

Since 1997, an emergent fungal disease named lethargic crab disease (LCD) has decimated stocks of the edible mangrove land crab Ucides cordatus (Linnaeus, 1763) (Brachyura: Ocypodidae) along the Brazilian coast, threatening the mangrove ecosystem and causing socioeconomic impacts. Evidence from a variety of sources suggests that the black yeast Exophiala cancerae (Herpotrichiellaceae, Chaetothyriales) has been responsible for such epizootic events. Based on the spatiotemporal patterns of the LCD outbreaks, the well-established surface ocean currents, and the range of ecological traits of Exophiala spp., a marine dispersal hypothesis may be proposed. Using in vitro experiments, we tested the survival and growth of E. cancerae CBS 120420 in a broad combination of salinities, temperatures, and exposure times. While variation in salinity did not significantly affect the growth of colony-forming units (CFUs) (P>0.05), long exposure times visibly influenced an increase in CFUs growth (P<0.05). However, higher temperature (30°C) caused a reduction of about 1.2-fold in CFUs growth (P<0.05). This result suggests that sea surface temperatures either above or below the optimum growth range of E. cancerae could play a key role in the apparent north-south limits in the geographical distribution of LCD outbreaks. In light of our results, we conclude that a fundamental step toward the understanding of LCD epidemiological dynamics should comprise a systematic screening of E. cancerae in estuarine and coastal waters.

Curcumin derivative C817 inhibits proliferation of imatinib-resistant chronic myeloid leukemia cells with wild-type or mutant Bcr-Abl in vitro

To find new kinase inhibitors that overcome the imatinib resistance in treatment of chronic myeloid leukemia (CML), we synthesized C817, a novel derivative of curcumin, and tested its activities against wild-type (WT) and imatinib-resistant mutant Abl kinases, as well as in imatinib-sensitive and resistant CML cells in vitro. 32D cells harboring WT or mutant Abl kinases (nucleotide binding P-loop mutants Q252H, Y253F, and imatinib contact residue mutant T315I), as well as K562/G01 cells (with whole Bcr-Abl gene amplication) were tested. Kinase activity was measured using Kinase-Glo Luminescent Kinase Assay Platform in recombinant WT and mutant (Q252H, Y253F, and T315I) Abl kinases. Cell proliferation and apoptosis were examined using MTT assay and flow cytometry, respectively. The phosphorylation levels of Bcr-Abl initiated signaling proteins were analyzed using Western blotting. Colony forming units (CFU) growth and long term culture-initiating cells (LTC-ICs) were used to test the effects of C817 on human leukemia progenitor/stem cells. C817 potently inhibited both WT and mutant (Q252H, Y253F, and T315I) Abl kinase activities in a non-ATP competitive manner with the values of IC₅₀ at low nanomole levels. In consistent with above results, C817 suppressed the growth of both imatinib-sensitive and resistant CML cells, including wild-type K562, K562/G01, 32D-T315I, 32D-Q252H, and 32D-Y253F cells with the values of IC₅₀ at low micromole levels. C817 (0.5 or 1 μmol/L) dose-dependently inhibited the phosphorylation of Bcr-Abl and downstream proteins STAT-5 and CrkL in imatinib-resistant K562/G01 cells. Furthermore, C817 significantly suppressed CFU growth and LTC-ICs, implicating that C817 could eradiate human leukemia progenitor/stem cells. C817 is a promising compound for treatment of CML patients with Bcr-Abl kinase domain mutations that confer imatinib resistance.

Universal Intravenous Access Cleaning Device Fails to Sterilize Stopcocks

Contamination of a central venous catheter may occur through use of conventional open-lumen stopcock devices (COLDs), or disinfectable, needleless, closed connectors (DNCCs). We investigated the effectiveness of a new universal IV access cleaning device (Site-Scrub) compared with 70% isopropyl alcohol prep pads for sanitizing COLDs or DNCCs inoculated with common catheter-associated pathogens. Site-Scrub was compared with 70% alcohol prep pads for sanitizing contaminated female Luer lock COLD or DNCC filled with sterile saline or propofol and 2 common bacterial central venous catheter contaminants (Staphylococcus epidermidis or Pseudomonas aeruginosa). Devices were contaminated using a glove touch (COLD and DNCC) or syringe tip (COLD). The primary end point of the study was colony-forming units (CFU) after 24 hours. The use of glove touch contamination, the contaminants, S epidermidis and P aeruginosa, produced CFU in saline-filled COLDs treated with the Site-Scrub, but not in those treated with alcohol pads (P < 0.001). Similar results were observed with propofol-filled COLDs (P < 0.001). For DNCCs filled with saline or propofol, both alcohol and Site-Scrub effectively reduced CFU growth compared with contaminated controls (P < 0.001). When COLDs were contaminated by treated syringe tips, there was no significant evidence of reduction in CFU growth by using either alcohol pads or Site-Scrub compared with contaminated controls. These data suggest that when the inner surface of the COLD is contaminated, both alcohol pads and Site-Scrub were not significantly effective in decontaminating the COLD. When the COLD rim is contaminated, however, alcohol pads outperform Site-Scrub. DNCCs were uniformly decontaminated with either treatment. Future work should focus on better access systems because current COLDs are difficult to decontaminate.

Noonan Syndrome Peripheral Blood Cells Show Enhanced Spontaneous Colonies Growth In Vitro and Hyperactive JAK and RAS Intracellular Signalling Pathways

Noonan Syndrome (NS) is an autosomal dominant congenital disorder characterized by several defects at births. In addition, children with NS show splenomegaly and monocytosis, and are predisposed to develop myelo proliferative disorders (MPD) which may resolve spontaneously or progress in an aggressive clinical course similar to Juvenile Myelomonocytic Leukemia (JMML). Somatic PTPN11 mutations, which encodes the protein tyrosine phosphatase Shp2, affect around 35% of patients with JMML, while germline PTPN11 mutations cause around 50% of NS cases (Cratz et al. Blood, 2005). Moreover, mutations in other genes affecting the RAS/RAF/ERK pathway, such as SOS1 and KRAS, have been identified in PTPN11-negative NS patients (Tartaglia et al. Nature Genetics, 2007). Yet, both JMML and NS/MPD patients show hypersensitivity to GM-CSF and spontaneous colonies growth (Lavin et al. Pediatric Blood Cancer, 2008). These evidences prompted the hypothesis that children with NS could maintain an abnormal status of hematopoiesis affecting in particular the myelomonocytic lineage.We studied 23 children with NS and 16 age-matched children with other genetic defect, affecting neither JAK-STAT nor RAS/RAF/ERK pathways. Peripheral blood (PB) cells were cultured in vitro in order to evaluate the spontaneous and factor-stimulated growth of colony-forming units. In addition, PB monocytes were evaluated for IL-6 (50 ng/mL)-induced response of pSTAT3, and GM-CSF-induced response of pSTAT5 and pERK (10 and 0.1 ng/mL respectively), measured by phosphoflow technique, as previously described (Gaipa et al, Leukemia, 2008).We observed a significantly enhanced in vitro spontaneous growth of erythroid colony-forming units (BFU-E) in NS (n=15) as compared to control subjects (n=10) (mean ± SD: 79.67% ± 30.15% vs 22.60% ± 23.00%, respectively; p=0.0004), and an augmented growth of granulocyte-macrophage units (CFU-GM), although not significantly (19.13% ± 17.20% vs 10.90% ± 17.72%; p>0.05). This enhanced capacity of NS cells to promote colony formation was confirmed also in the presence of growth factors in the medium.Phosphoflow assay showed an enhanced amount of stimulated pSTAT3 and pSTAT5 positive cells in NS patients as compared to control subjects (pSTAT3: 41.01% ± 32.97% (n=18) vs 22.99% ± 22.38% (n=13); p=ns, and pSTAT5: 98.20% ± 2.55% (n=5) vs 63.40% ± 40.17% (n=5), p<0.05), Interestingly, preliminary experiments showed that NS patients also have higher rate of pERK response upon stimulation with GM-CSF.In summary, we demonstrated that children with NS, but without MPD, do have significantly enhanced capacity of both BFU-E and CFU-GM units formation in vitro. GM-CSF hypersensitivity has been demonstrated in NS/MPD (Lavin et al. Pediatric Blood Cancer, 2008), thus supporting the observed enhanced growth of CFU-GM units in NS patients with persistent monocytosis. Intriguingly, abnormal growth of BFU-E was not previously reported in these patients. The biological significance of these enhanced growth capacities, particularly in absence of recognized MPD status, should be further investigated. Signalling in transduction pathways affected by Shp2 protein seems to be latently activated in NS, although these observations need to be fully validated with further experiments. Collectively, our findings open new opportunities to investigate the almost unknown hematological alterations existing in NS patients. In addition, they may lead to an enrichment of the laboratory tools potentially able to recognize signs of progression toward malignant MPD (including JMML) in children with NS. Disclosures:No relevant conflicts of interest to declare.

Viability of umbilical cord blood mononuclear cell subsets until 96 hours after collection

Umbilical cord blood (UCB) is a good source of hematopoietic stem cells for transplantation and cell therapy. In 2006, the Brazilian Public Network of Cord Blood Banks was founded; however, because our country is large, logistic problems could hamper the collection of numerous samples. Our aim was to evaluate the viability of several UCB cell subsets until 96 hours after collection, to examine whether this delay would be acceptable for processing and freezing the samples. Two experiments were performed: in the first one, volume reduction of the UCB units was carried out before analysis. In the second one, analysis was carried out with no previous manipulation. Samples were stored at room temperature and one aliquot was taken daily for analysis. We examined CD34+ cell, B-cell precursor, mature B and T lymphocyte, monocyte, granulocyte, and mesenchymal stem cell (MSCs) concentrations. Thirty-six UCB units were analyzed. CD34+ cells and mature T lymphocytes increased (viability 99%). Mature B lymphocytes and MSCs decreased, maintaining viability. Granulocytes decreased with loss of viability. Monocytes and immature B lymphocytes remained stable. Clonogenic assays showed a decrease in colony-forming unit (CFU) number in UCB units stored for 96 hours. UCB manipulation did not influence cell viability. All cell subsets remained viable until 96 hours after collection. CD34+ cells and T lymphocytes increased, probably due to the loss of other subsets. CFU growth during the period analyzed and confirmed stem cell functionality, despite the decrease at 96 hours. Results demonstrated that UCB units could probably be processed up to 96 hours after collection.

What Clinical Characteristics of Mothers and Babies Favorably Influence Cord Blood Potency? Defining Parameters That Can Guide Public Cord Blood Collection

Abstract 484 Background:Cord blood (CB) is a rich source of hematopoietic stem cells and provides access to transplantation for many patients who otherwise would not have a suitably matched donor. Numerous international public CB banks are actively accruing units to the public inventory. While public banking is an activity that benefits many patients by increasing access to hematopoietic stem cell transplantation, it is financially challenging to maintain operations of a public bank which must front all costs of inventory building and overall operations. It is well recognized that total nucleated cell (TNC) content as well as numbers of CD34+ and colony forming unit (CFU) cells define a superior cord blood unit (CBU). In the US, criteria defining a higher quality CBU have been established through the C.W. Bill Young Cell Transplantation Program’s National Cord Blood Inventory. Applying current criteria, only ∼40% of collected CBUs meet criteria for banking. To improve the yield of “bankable” CBUs and allow for better focus of resources, we hypothesized that certain maternal/donor characteristics may be predictive of TNC (also, CD34+ or CFU) and, therefore, be more likely to meet banking criteria. Methods:Between 9/07–7/09, CBUs donated to the Carolinas Cord Blood Bank (n=5282) and meeting criteria for public banking (n=5268) or identified for directed donation (n=14) were analyzed. Standard criteria for donor eligibility were followed, including gestational age >34 weeks, uncomplicated pregnancy and delivery, singleton birth, and maternal age ≥18 years. Technical characteristics were obtained as part of routine banking procedures (including post-processing TNC, CD34+ and CFU) and were correlated with clinical features of the mother and baby including maternal age, delivery type, baby gender, birth weight (BW), gestational age, race/ethnicity and collected CB volume. Univariate and multivariate logistic regression models were created dichotomizing for TNC, CD34+ and CFU content. Univariate and adjusted odds ratios are provided. Results:In this study cohort, the infant donors had a median gestational age and BW of 39.0 weeks (range, 34–42 weeks) and 3580g (range, 1800–5724g), respectively. Both genders were evenly represented although there were more Caucasian infants (58.9%) followed by African American (20.2%) and Hispanic (12.9%). Slightly more infants were born via vaginal delivery (59.8%) to mothers with a median age of 29 years (range, 18–53 years). The median TNC count was 11.7×108 (range, 2.9–55.5), CD34+ cells were 3.36×106 (range, 0.017–98.2) and CFU content was 34.0×105 (range, 0.6–193.3). In univariate analysis, maternal/donor characteristics that were associated with high TNC (defined as a TNC of >10×108) included Caucasian race [OR 1.29 (95%CI 1.14–1.46) p<0.0001], Caesarian delivery [OR 1.23 (95%CI 1.09–1.39) p<0.0001], higher gestational age [OR 1.16 (95%CI 1.10–1.22) p<0.0001] and BW>3500g [OR 1.86 (1.64–2.09) p<0.0001]. Conversely, African American race was a negative predictor of high TNC [OR 0.76 (95%CI 0.66–0.88) p=0.0002] while gender and Hispanic race were not significant. In multivariate modeling of TNC including maternal/donor characteristics, BW [>3500g; OR 1.75(95%CI 1.54–1.98) p<0.0001] was the best predictor of a high TNC followed by Caucasian race [OR 1.20(95%CI 1.06–1.36) p=0.004] and Caesarian delivery [OR 1.20 (95%CI 1.05–1.36) p=0.006). We also examined clinical variables predictive of a high CD34+ content (defined as >3×106). In univariate analysis, male gender [OR 1.31 (95%CI 1.19–1.49) p<0.0001], BW>4000g [OR 1.93 (95%CI 1.66–2.25) p<0.0001], older gestational age [(OR 1.19 (95% CI 1.14–1.25) p<0.0001] and Caucasian race [OR 1.18 (95%CI 1.06–1.32) p=0.003] were predictive of high CD34+ content. Interestingly, neither maternal nor donor characteristics were associated with high CFU growth in univariate or multivariate analysis. Conclusions:Maternal/donor characteristics are predictive of TNC and CD34+ in collected CBUs. However, no clinical characteristic predicted graft CFU content, the primary determinant of CBU potency. These associations could be used to prioritize donations, optimizing resource utilization and financial modeling. Of note, African American race was associated with lower TNC indicating that increased numbers of African American CBUs will need to be collected to achieve a robust inventory of higher quality units. Disclosures:No relevant conflicts of interest to declare.

10% Povidone-Iodine May Be a Practical Field Water Disinfectant

A paucity of data exists regarding the use of iodophores such as povidone-iodine (PVI) to disinfect water. We sought to determine a practical minimal disinfecting concentration of 10% PVI over different contact times and temperatures when added to water inoculated with E. coli. 1:100, 1:1,000, and 1:10,000 dilutions of 10% PVI were created. Escherichia coli was exposed to these dilutions for 5, 15, and 30 minutes at 10, 20, and 30°C. Bactericidal activity was neutralized with 0.5% sodium thiosulfate. Mean viable colony forming units (CFUs) was determined after triplicate plating on Luria-bertani agar and 24 hours of incubation at 37°C. Effective bactericidal activity was defined as a 5-log reduction. Of the 200,000 E. coli plated, no CFUs were observed after exposure to the 1:100 dilution. After 5 minutes of contact time with the 1:1,000 dilution, at 10°C CFUs were too numerous to count (TNTC), at 20°C the mean CFU count was 92 (standard error ±11), and at 30°C the mean CFU count was 25 (standard error ±8). No CFUs were observed after 15 minutes of exposure to the 1:1,000 dilution across experimental temperatures. The 1:10,000 dilution always yielded CFU growth that was TNTC. The lowest disinfecting concentration of 10% PVI was the 1:1,000 dilution at 15 minutes of contact time. This supports the use of PVI for water disinfection against E. coli, the organism most commonly responsible for traveler's diarrhea. Further studies may assess its effectiveness against more virulent water borne pathogens.

Colony Forming Unit (CFU) Assay with High Resolution Digital Imaging (HRDI): a Reliable, Standardized Technology for Evaluation of Cord Blood Potency Before and After Cryopreservation.

Abstract 2160Poster Board II-137 Background:The Total Nucleated Cell dose (TNC) is the graft characteristic most commonly used as indicator of engraftment potential for CB unit selection. Even thought well standardized, TNC does not reflect the functional state of hematopoietic progenitor cells, an important determinant of their engraftment ability, and therefore, of the CB unit quality. In contrast, CFU is a better graft characteristic since it measures the functional state, repopulation capacity and number of hematopoietic progenitor cells of a CB graft. However, it has not yet been standardized, so that it can be used for comparison of CB unit quality among different CB banks, or for evaluation of cryopreserved units prior to release for transplantation. There is therefore, an urgent need of a potency assay that could allow transplant physicians to select the highest quality unit for their patients.We sought to evaluate whether our new technology that combines the CFU assay with High Resolution Digital Imaging and colony staining (CFU-HRDI) [ASH 2008;2306], would allow for a better standardization of the traditional CFU assay and the testing of a large number of CB samples in a ”High Throughput” approach. Further, we examined whether the CFU-HRDI can be a reliable method for determination of CBU quality before release for transplantation. Methods and Results:CFU growth was assessed in a total of 7,447 CBU over a period of 14 months, by using the HRDI technology. Samples were obtained after CB processing (AXP® system) and all were evaluated in duplicates. The HRDI system achieves a resolution of 7.6 μM per pixel allowing a clear view of all colonies in the dish with their barcoded IDs. A short one-step colony staining procedure provides an even better definition of CFU-GM/E and CFU-GM by bestowing a specific color on each type. A good correlation was observed after comparison of the CFU-HRDI against traditional microscopy enumeration (R2= 0.95; p<0.001). The number of CFU (average: 3109 10×3 +/- 2100 CFU/CBU) correlated strongly with total mononuclear cell count-MNC (R2=0.66, p<0.001); TNC (R2=0.64, p<0.001), nucleated red blood cells-NRBC (R2=0.44, p<0.001) and especially with CD34+ cell content (R2=0.81, p<0.001) of the CBU.To evaluate the reproducibility obtained by the use of HRDI, CB samples (n=3) were plated multiple times each (n=24 times) and five operators with different levels of expertise counted and classified CFU in all dishes. CFU-HRDI allowed excellent reproducibility for counting and classification (CFU-GM and CFU-Mixed-Red; R2≥0.9; p<0.001; n=72). Importantly, correlation was good even when HRDI showed CFU growth of several sizes and shapes for different CB units, a feature that increases the complexity of CFU classification and enumeration. Furthermore, to evaluate the effect of cryopreservation on CB quality, a total of 186 attached segments were assessed after thawing for CFU, CD34 and viability (7-AAD). Among those units, n=43 were split in two bags after processing and thus, the respective segments could be compared (R2=0.70, p<0.001). All segments showed good viability (95 +/- 5%; range 71-100). Additionally, the CFU and CD34 cell content of the unit pre-cryopreservation were well correlated when compared to the results of their respective segments (R2= 0.57, p<0.001 and R2= 0.59, p<0.001 respectively; n=58). Summary:Our new strategy (CFU-HRDI) shows reliability and reproducibility and importantly, supports high-throughput implementation as shown by the assessment of more than 7,000 CBU/year. CFU can be objectively classified and enumerated and thus CFU-HRDI can be standardized to be used as a potency determinant of CBU. Digital images can be stored for future review or further classification of CFU colonies if needed for their association with transplant engraftment. The feasibility of CFU growth testing from thawed segments and its correlation with post-processing CFU counts strengthens its use as a practical quality control measure for the cryopreservation process prior to a CBU release for transplantation. Disclosures:No relevant conflicts of interest to declare.

Clonally Related B-NHL Populations and Cell Lines Harbor Subsets with Significant Phenotypic and Functional Heterogeneity.

Abstract 1699Poster Board I-725Many malignancies such as myeloid leukemia, breast, colon, and CNS tumors appear to harbor phenotypically and functionally distinct populations of cells with self-renewal, differentiative, and tumorigenic capacity. These unique populations, influencing tumor growth and persistence, have been termed “cancer stem cells,” or “tumor-initiating cells.” No such tumor-initiating capacity has been prospectively identified in non-Hodgkin lymphoma (NHL). Identification of these novel sub-populations in B-NHL is particularly challenging given the unique proliferative capacity, differentiation potential, and population diversity of the peripheral B cell subsets from which lymphomas arise. Our studies have focused on the identification of sub-populations that meet criteria for “lymphoma stem cells” (LySC) in B-NHL samples. Two experimental sources of NHL cells were used: i) primary human DLBCL (TXN190, TXN131, C10, C12) derived at our institution from malignant effusions, and, ii) continuously cultured, widely used DLBCL lines (OCI-Ly-7, -10, -19).Using multiparameter FACS, we initially performed immunophenotypic studies to investigate a broad range of cell surface markers. Notably, we observed significant heterogeneity in the surface antigen density for CD22, CD27, CD45R/B220, and CXCR5. These data suggest that subpopulations residing within NHL populations may possess functional heterogeneity. To test this concept, we first performed colony-forming unit (CFU) assays in methycellulose medium. Analysis of 7 specimens shows a CFU frequency ranging from .0003% to 10% of the overall population, demonstrating functional heterogeneity with regard to in vitro colony-forming ability. Surprisingly, we found that generation and successful re-plating of B-NHL colonies did not require the addition of exogenous factors such as PHA-conditioned medium, cytokine, or interleukin supplementation. Next, we performed flow cytometric separation of candidate subpopulations as a means of segregating functional potential. These studies showed that CD45R/B220 expression levels appear to correlate with colony-forming ability. B-NHL cell fractions with lower levels of expression of the murine pan-B cell marker CD45R/B220 appear to be enriched for CFU activity in methylcellulose culture compared to B220-high cells. In addition, CD45R/B220-low B-NHL has higher tumorigenic potential (assessed by kinetics of growth rate) in assays of subcutaneous tumor nodule generation. These studies indicate differential potential of NHL subpopulations, and may begin to establish a phenotype of LySC. Finally, we measured in vitro clonogenic potential using two assays – i) limiting dilution suspension culture, and - ii) CFU assays. Interestingly, the results from these two independent assays do not correlate well in B-NHL; clonogenic potential appears to be substantially higher in limiting-dilution suspension cultures when compared to methylcellulose assays. These findings indicate that the relationship between CFU frequency and growth potential in B-NHL populations may differ from those found in normal hematopoietic cell types, and suggest that some caution should be exercised in the interpretation of B-NHL CFU assays.The adaptation of assays useful for identification of tumor-initiating sub-populations has also been useful in defining subsets of clonally related B-NHL cells that appear immunophenotypically and functionally distinct in vitro and in vivo. Our studies support the notion that B-NHL tumor-initiating cells may comprise a unique population amenable to prospective immunophenotypic and/or functional identification. Implicit in the concept of these putative LySC is that the control, and ultimately the eradication, of such unique NHL populations may result in improved clinical outcomes.[SAW and RMR contributed equally to this work. Funded by the Lymphoma SPORE, P50 CA130805501A1, PI: Richard Fisher, MD.] DisclosuresNo relevant conflicts of interest to declare.

Hematopoietic Colony Forming Unit: Development of a High-Throughput CFU Assay Strategy by the Use of High-Resolution Digital Images Stored in a Laboratory Information System.

Background: Umbilical Cord Blood (CB) is an increasingly accepted graft source for patients lacking related donors. Graft characteristics currently used as determinants of quality and engraftment potential of CB units include the enumeration of Total Nucleated Cell (TNC), CD34+ and Colony Forming Units (CFU). Among those, the 14 day-CFU assay is the only one that determines the functional state as well as the number of hematopoietic progenitor cells. Thus, CFU in pre and post cryopreservation/thawing specimens are strong independent predictors of CB graft engraftment. Traditionally, evaluation of CFU growth is performed by light microscopy (traditional classification), is time consuming, subjective and difficult to standardize.Aims: A) To evaluate whether the incorporation of high-resolution digital imaging and colony staining could make reading the traditional CFU assay objective and readily standardizable. B) To validate the new strategy vs traditional classification. C) To develop a computer based laboratory information management system (LIMS) to support high-throughput CFU assay.Methods and Results: After 14 days of CB culture (CFU assay-Stem Cell Technologies), an image of the 35 mm culture dishes was captured using a high-resolution photographic camera based digital imaging system, which achieves a resolution of 7.6 μM per pixel and thus, allows a clear view of all colonies in the dish with their barcoded IDs. A short one-step staining with MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) allows an even better definition of CFU-GM/E; CFU-GM and CFU-E by bestowing a specific color on each type (dark purple, purple and red respectively) against a uniformly clear background. A good correlation was observed after comparison of the new strategy against traditional enumeration (R2 linear= 0.95; n= 122 culture dishes evaluated). Low variation was observed after 151 cultures were independently classified and enumerated by three different operators (CV%= 8.9; range 1–27%). Sample plating introduced variation of the CFU assay, in an experiment where nine CB samples were evaluated by multiple plating (Intra-assay CV%= 21.9 %; range 3.4–34.5% and Inter-assay CV%= 23.3%; range 12.6–35%). A computer based laboratory information (LIMS) was developed to store all culture dishes, linked by unique barcoded ID labels to a specific CB unit, and including CB image, incubator location, plating and counting dates, as well as detailed colony enumeration. This system has been used for 5 months in our laboratory and more than 3,000 CB units have been tested in duplicate (average: 30 CB/day). Images of an average of 60 culture dishes and MTT staining can be performed in less than two hours. The specific coloration of CFU colonies allows faster classification and enumeration and thus, permits a more precise analysis of CFU colonies and its relation with CD34+ cell content and post-transplant engraftment.Summary: With this new strategy, CFU can be objectively visualized, differentiated and counted; the digital images can be stored for future review and refined classification. The described system provides computerized information on optical assay parameters and has become an invaluable tool supporting high-throughput implementation. Thus, the combination of high resolution imaging, one-step staining and the traditional CFU assay overcome most technical challenges of the conventional method, supporting standardization and yielding high reproducibility to the assay which can be easily implemented in CB banks where large numbers of samples need to be tested daily.