The role of the dehydration stage in the post-hypertonic hemolysis of mammalian erythrocytes

The aim of this study was to assess the level of damage to mammalian erythrocytes under post-hypertonic shock depending on the concentration of NaCl in the dehydration medium and to determine the effect of hypertonic NaCl solutions on the condition of mammalian erythrocytes by flow cytometry. To achieve this goal, spectrophotometric and cytometry research methods were used. The data obtained showed that post-hypertonic lysis of mammalian erythrocytes depends on the concentration of NaCl in the dehydration medium. The most sensitive to the effects of post-hypertonic shock are rat erythrocytes, the least sensitive are rabbit cells. Cytometry studies revealed significant changes in the histograms of the distribution of erythrocytes of all mammalian species with increasing salt concentration in the dehydration medium. These changes are species-specific and are probably related to changes in cell volume and morphology. The data revealed a relationship between the level of post-hypertonic hemolysis and the values of such indicators as the median distribution and the coefficient of variation. Thus, an increase in the sensitivity of mammalian erythrocytes to post-hypertonic shock with increasing salt concentration in dehydration medium was usually accompanied by a decrease in the median cell division, and higher values of the coefficient of variation are characteristic of mammalian erythrocytes resistant to post-hypertonic shock.

Characterization of potassium, sodium and their interactions effects in yeasts

Biotechnology requires efficient microbial cell factories. The budding yeast Saccharomyces cerevisiae is an important cell factory but for a sustainable use of natural resources more diverse cellular attributes are essential. Here, we benchmarked non-conventional yeasts Kluyveromyces marxianus (KM) and Rhodotorula toruloides (RT) against the extensively characterized strains of S. cerevisiae, CEN.PK and W303. We developed a computational method for the characterization of cell/vacuole volumes and observed an inverse relationship between the maximal growth rate and the median cell volume that was responsive to monovalent cations. We found that the supplementation of certain K+ concentrations to CEN.PK cultures containing 1.0 M Na+ increased the specific growth rate by four-fold with a parabolic shift in the median cell/vacuole volumes. The impairment of ethanol and acetate utilization in CEN.PK, acetate in W303, at the higher K+/Na+ concentrations implied an interference in the metabolic pathways required for their consumption. In RT cultures, the supplementation of K+/Na+ induced a trade-off in glucose utilization but alleviated cellular aggregates formation where specified cationic concentrations increased the beta-carotene yield by 60% compared with the reference. Our comparative analysis of cell/vacuole volumes using exponential phase cultures showed that the median volumes decreased the most for KM and the least for RT in response to studied cations. Noteworthy for the implication in aging research using yeasts, the vacuole to cell volume ratio increased with the increase in cell volume for W303 and KM, but not for CEN.PK and RT.ImportanceFor designing efficient bioprocesses characterization of microbial cell factories in the relevant culture environment is important. The control of cell volume in response to salt stress is crucial for the productivity of microbial cell factories. We developed an open source computational method for the analysis of optical microscopy images that allowed us to quantify changes in cell/vacuole volumes in response to common salts in yeasts. Our study provides a framework for appreciating the role of cellular/organellar volumes in response to changing physiological environment. Our analysis showed that K+/Na+ interactions could be used for improving the cellular fitness of CEN.PK and increasing the productivity of beta-carotene in R. toruloides, which is a commercially important antioxidant and a valuable additive in foods.

Two new species of the genus Givarbela Clench, 1957 (Lepidoptera: Cossidae: Hypoptinae) from South Neotropics

Givarbela steinbachi was described as a new genus and species by Clench (1957), based on 21 specimens from central Bolivia (Prov. del Sara; Buena Vista and Rio Japacani). He indicated that Givarbela (Figs 1‒8) belongs to the Langsdorfia-Givira group of genera but differs from them by the following combination of characters: “R2 stalked with R3-R4, R5 free; the long palpi; absence of fore tibial epiphysis; absence of median cell-vein on fore wing and the open cell-end there; stalked M2-M3 on hind wing; absence of all but traces of a single hind wing anal vein; short hind wing cell; deeply excised hind wing costa“ (Clench 1957).

Performance of Biocept's sample collection for tumor cell analysis.

e23036 Background: Liquid biopsy is a minimally invasive and cost effective way to assess cancer biomarkers without the risk of surgical biopsy complications. Circulating tumor cell (CTC) analysis from body fluids can provide critical information towards early detection, prognosis and treatment decisions. Accurate CTC evaluations require optimal cell preservation. Cell lysis, DNA degradation, or membrane alterations compromise CTC analyses and accurate diagnoses. This work compares Biocept’s proprietary CEE-Sure BCT and Saccomanno's Cytology Fixative largely used for sputum collection. Methods: One million BT474 (HER2 amplified) or H3112 (ALK re-arranged) cells were spiked into 500 µl medium; 500 µl of CEE-Sure or Saccomanno fixative was added. Tubes were stored at 4°C for 1 day, 1 week, or 1 month. Cells were centrifuged, resuspended, and counted (Celigo). Around 150 cells in 15 µl of RPMI medium were flowed into Biocept's microfludic system for cell capture; recovery (%) was calculated. Captured cells were subjected to fluorescent in situ hybridization (FISH) analyses for qualitative signal evaluation. Results: As similar results were observed for both cell lines and all time points, combined data will be shown. Median cell recovery after CEE-Sure incubation was 14.1% (range 1.7–44%, n = 12) vs 5.4% (range 0.07–26.9%, n = 12) in Saccomanno's fixative. Median cell capture of ~150 cells fed into Biocept’s microchannel was 96% (range 72-98%) for CEE-Sure vs 82% (range 21-96%) for Saccomanno. Paired t-tests showed significant differences for both recovery and capture. FISH signals from CEE-Sure samples were qualitatively rated Fair to Good, while Saccomanno samples had Poor to Fair, grainy, non-specific signals. Conclusions: This preliminary work shows consistently higher cell recovery, better cell membrane maintenance, and higher quality FISH signals for samples stored in Biocept's CEE-Sure vs Saccomano’s fixative. With liquid biopsy testing gaining rapid traction, maximal cell stability during the transport and storage are crucial. Additional fixative comparison is ongoing in various patient specimen types. These results support expansion of molecular analyses in sputum samples enriched for lung epithelial cells.

Haploidentical Allogeneic Transplantation As Salvage in Relapsed Multiple Myeloma

Allogeneic stem cell transplantation (SCT) using HLA-haploidentical (HAPLO) related donors has been an effective strategy for treating hematologic malignancies in patients who lack a matched donor. In multiple myeloma (MM), allogeneic SCT used in tandem or as salvage therapy can be a potentially curative option, however, there is little data regarding the use of HAPLO donors in this population. We report 10 patients with MM who received HAPLO allografts between 2011-2014. Median age was 53 (range 28-61) and 5 patients were male. All patients, except one, were relapsed or refractory to at least 1 prior autograft. Patients with pre-transplant CD4 counts >200/uL received a fludarabine (Flu)-based regimen for in-vivo lymphodepletion to prevent graft rejection. Conditioning regimen for transplantation was Flu 30 mg/m2 daily x5 with cyclophosphamide (Cy) 14.5 mg/kg daily x 2, followed by TBI 200-400 cGY in 1 or 2 fractions. Post-transplant immunosuppression consisted of Cy 50-60 mg/kg on days +3 and +4, followed by tacrolimus and mycophenolate starting on day +5. All patients received filgrastim starting on day +5. Six patients received bone marrow (BM) with a median cell dose of 2.76 x 10e6 CD34cells/kg (range 1.1-4.02) and 4 patients received peripheral blood stem cells (PBSC) with a median cell dose 4.01 x 10e6 CD34 cells/kg (range 1.05-4.04). One patient developed sepsis on day +14 and died on day +31 without hematologic recovery. All other patients achieved engraftment, with median neutrophil engraftment of 18 days (range 14-45) and median platelet engraftment of 21 days (range 14-53). There was no difference between engraftment for BM or PBSC. Median time to neutrophil engraftment was 17 days for BM and 19.5 days for PBSC (p=ns). Median time to platelet engraftment was 29 days for BM and 20.5 days for PBSC(p=ns). Median time to full donor chimerism was 32 days overall (range 27-61); 38 days for BM (range 29-61) and 27.5 days for PBSC (range 27-32). At a median follow up of 10 months (range 1-35), 6 patients are alive. Of those, 2 patients are in CR, 3 in VGPR, and 1 with stable disease (SD). The 2 year overall survival is 46%. The causes of death for 4 patients were sepsis (1), progressive disease (2), and chronic inflammatory demyelinating polyneuropathy (CIDP) (1). Eight patients developed acute graft versus host disease (GVHD), with one having grade III, and 7 with grade I-II. Five patients were treated for chronic GVHD, 3 mild and 2 moderate by NIH criteria. None of the deceased patients developed chronic GHVD and all of the surviving patients had chronic GVHD, except for 1 who is prior to day +100 at the time of this report. Four patients were treated for grade 3 infections, with 1 infection-related death. Five patients relapsed or progressed their disease after HAPLO transplant, with a median time to progression of 7.8 months (range 3.2-11.4). Of these, 1 is in VGPR after salvage therapy, 1 has SD after salvage therapy and donor lymphocyte infusion, 2 died from relapse and 1died from CIDP. KIR typing was available for 8 recipient and donor pairs. There were 2 KIR B/x to A/A transplants, with1 patient deceased and 1 alive. All of the 4 KIR B/x to B/x recipients are alive, and the 2 KIR A/A to B/x recipients are deceased. This small series suggests HAPLO transplantation can be used in patients with advanced MM who are without a suitable matched donor. Our patients achieved timely hematologic engraftment and donor chimerism with BM or PBSC. There was no fatal acute GVHD, and as previously reported, chronic GVHD may be protective against relapse. Further studies need to be done to further elucidate the impact of KIR typing on transplant outcomes. Overall Survival Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Morphology Study of Four Kinds of Syrphid

This research was mainly on morphology observation of syrphid common species including Episyrphus balteatus, Eristalis latréillè, Eupeodes osten, and Melanostoma schiner. Observations of four species of syrphid morphological characteristics in Shenyang were of the first radial cell area (R1), the third radial cell area (R3), the fifth radial cell area (R5), the second median cell area (M2), the first anal cell area (1A), spurious vein and wing length. The results showed that the first radial cells area, the third radial cells area, the fifth radial cell area, the second median cells area, the first cell area and wing length of M. schiner were the minimum, and spurious vein length was the maximum. The first radial cell area of E. latréillè was larger than that in other three species. The first radial cell area of E. latréillè was larger than that in other three species. The third radial cell area, the fifth radial cell area and the second median cell area of E. balteatus were larger than that in other three species. The wing length of E. osten was longer than that in other three species. Significance test showed that syrphid wings of the same individual from the four species had no significant differences.

Extent of neutrophil (NTP) infiltration in benign lymph nodes (LNs) to predict survival in patients with muscle-invasive bladder cancer (MIBC).

e15522 Background: In preclinical models, NTPs appear to establish a pre-metastatic niche that fosters the invasion of metastases (Kowanetz et al. PNAS 2010). This observation still requires clinical validation in MIBC. Methods: Benign LN tissue was obtained from patients (pts) who had undergone cystectomy and LN dissection for documented MIBC. Immunohistochemical (IHC) staining for CD15 (a NTP marker) was performed. Interleukin-17 (IL-17) and phosphorylated signal transducer and activator of transcription 3 (pSTAT3), putative mediators of NTP recruitment, were assessed through the same method (Laan et al. J Immunol 1999; Fielding et al. J Immunol 2008). Positively staining cells were counted and averaged over 8 high power fields. Pts were stratified by the median cell count for each biomarker. Analyses of overall survival (OS) were performed using the Kaplan-Meier method and log-rank test. Results: Of 55 pts with MIBC, 19 pts received no neoadjuvant chemotherapy (NAC), while 36 pts had received NAC with either GC (n=17) or MVAC (n=19). CD15 and IL-17 expression was significantly lower in pts with prior NAC (P<0.001 for both), while expression of pSTAT3 was similar in both groups. Furthermore, across the whole cohort, a strong association was seen between expression of CD15 and IL-17 (r=0.73, P<0.001). Amongst patients with no prior NAC, median OS was higher in those pts with low CD15 v high CD15 (158.7 mos v 36.9 mos, P=0.02), and low pSTAT3 v high pSTAT3 (NR v 106.4 mos, P=0.04). Median OS was numerically higher in pts with low IL-17 v high IL-17 (114.5 v 36.9 mos; P=0.14). Patients with both low CD15/IL-17 had a particularly favorable outcome. Amongst pts with prior exposure to NAC, no difference in survival was noted based on CD15, pSTAT3 or IL-17. Conclusions: NTP recruitment to benign LNs (potentially mediated by IL-17) may be prognostic of OS in pts with MIBC who have not received NAC. Bolstered by these findings, the prognostic value of NTP recruitment will be examined prospectively in SWOG 1011, a trial comparing limited v extended LN dissection in pts with MIBC.

Stem Cell Mobilization in Poor Mobilizers Using Plerixafor and G-CSF with or without Chemotherapy: A European Perspective

Abstract 2993 High-dose chemotherapy followed by autologous stem cell transplantation is an approved therapeutic intervention in relapsed Hodgkin-lymphoma (HL) and Non-Hodgkin lymphoma (NHL). In multiple myeloma (MM) it remains standard of care in first remission. Unfortunately, a significant portion of patients fail to mobilize and collect a sufficient amount of hematopoietic stem cells, being considered as “poor-mobilizers”. The effectiveness of the hematopoietic stem cell mobilizing agent plerixafor was evaluated in nationwide compassionate use programs in 13 European countries and reported to the European Consortium of Stem Cell Mobilization (ECOSM). Here we describe the mobilization success of 580 proven poor-mobilizers (304 male, 276 female) with NHL, HL and MM in Europe between May 2008 and August 2009. Furthermore, we analyzed the mobilization of stem cells in major NHL subgroups. All patients received plerixafor plus granulocyte colony-stimulating factor in standard doses with or without chemotherapy. Two-hundred seventy patients with NHL (138 male, 132 female) with a median age of 56 years (range 12 – 75 years) and a median of two prior chemotherapy regimens (range 0 – 10) were enrolled. Median cell yield was 2.56 × 10 ^6 CD34+ cells/kg BW (range 0 – 17.37). The general accepted minimum of 2.0 × 10 ^6 CD34+ cells / kg bodyweight (BW) for transplantation was reached by 175 patients (64.8%) in a median of two apheresis sessions (range 1 – 4). Thirty-four patients (12.6%) yielded more than 5.0 × 10 ^6 CD34+ cells/kg BW. There were no significant differences in in stem cell harvests regarding number of prior mobilization attempts or number of prior chemotherapeutic regimens, as well as in comparing patients with diffuse large B cell lymphoma (n=28), follicular lymphoma (n=15), and mantle cell lymphoma (n=24), respectively. Fifty-four HL patients (24 male, 30 female) with a median age of 36 years (range 19 – 76) and a median of three prior lines of therapy (range 1 – 5) were enrolled. Median cell yield was 3.14 × 10^6 CD34 cells/kg BW (range 0 – 32.6). Forty-four patients (81.5%) collected the minimum of 2.0 × 10^6 CD34+ cells/kg BW in a median of two apheresis sessions (range 1 – 4). Twelve patients (22.2%) collected more than 5.0 × 10 ^6 CD34+ cells/kg BW. A total of 256 patients (148 male, 108 female) with a median age of 60 years (range 28 – 76) diagnosed with MM were enrolled. Patients had received a median of two prior lines of treatment and collected a median of 3.60 × 10 ^6 CD34+ cells/kg BW (range 0 – 15.27) in a median of two apheresis sessions (range 1 – 5). The minimum of 2.0 × 10 ^6 CD34+ cells/kg BW was collected by 209 patients (81.6%). Eighty-two patients (32.0%) yielded more than 5.0 × 10 ^6 CD34+ cells/kg BW allowing tandem transplantation. Overall, the CD34+ cell yield was significantly higher in MM patients than in NHL patients (p <0.0001) and also significantly higher in HL patients than in NHL patients (p =0.013). CD34+ cell yield was not statistically significant between MM patients and HL patients. Furthermore, the number of patients collecting the minimum of 2.0 × 10 ^6 CD34+ cells/kg BW was significantly higher in MM patients compared to NHL patients (p <0.0001) and also significantly higher in HL compared to NHL patients (p =0.017). Analyzing the mobilization strategies and collection success of individual countries demonstrated only minor variations compared to the global results. Chemomobilization and steady state mobilization are used in most countries; however, there is a clear preference for chemotherapy combined with G-CSF/plerixafor in the Czech Republic, Germany, Hungary, Italy and Poland. The data emphasize the role of plerixafor in patients who failed prior mobilization attempts, but the development of improved strategies in poor mobilizers especially with NHL is required. Disclosures: Duarte: Genzyme: Membership on an entity's Board of Directors or advisory committees. Kröger:Genzyme: Membership on an entity's Board of Directors or advisory committees. Mohty:Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hübel:Genzyme: Membership on an entity's Board of Directors or advisory committees.

A pilot study of myeloablative (MA) autologous stem Cell (Auto SCT) followed by reduced intensity (RI) allogeneic transplantation (AlloSCT) in children with relapsed/refractory(R/R) Hodgkin’s disease (HD)

20007 Background: Allo SCT may benefit patients with R/R HD by providing a graft vs lymphoma effect. Peggs et al (Lancet 2005) demonstrated durable engraftment and reduced non relapse mortality (NRM) in HD pts post RI Allo SCT. Carella et al (JCO2000) and Gutman et al (BMT2005) demonstrated the success of MA Auto SCT followed by RI AlloSCT in adults with refractory lymphoma. We investigated the feasibility of MA Auto SCT followed by RI Allo SCT in children with R/R HD. Methods: MA conditioning prior to AutoSCT was CTX 1,500 mg/m2 x 4 d, BCNU 100 mg/m2 x 3d, VP-16 800 mg/m2 x 3d. AlloSCT conditioning was fludarabine 30 mg/m2 x 5d, busulfan 3.2 mg/kg x 2d, and R ATG 2 mg/kg x 4d (unrel. donor). CD20+ patients received rituximab (375 mg/m2/wk x4) and all pts received involved field radiotherapy (IFRT). Results: Ten pts have enrolled, 2 pts did not proceed (parental withdrawal) to RI AlloSCT (Donors: 1 MRD, 2 MUD, 5 UCB). Median time to RI AlloSCT after MA Auto SCT was 142 d (97–219). The median cell dose was 3.43 x 107 TNC/kg for UCB grafts (n=5). Engraftment was achieved at a median of 20.5 d for PMN and 46.5 d for PLT. Donor chimerism reached ≥ 95% in all pts by day 100 with a median follow up of 703d (128–2025). Toxicities were grade (3) hematuria (n=1), (3–4) infection (n=7), (4) pulmonary fibrosis (n=1), (4) hearing loss (n=1), (4) neurotoxicity (n=1). GVHD: grade II-III aGVHD (3/8), cGVHD (3/8). Six patients are alive and NED post allo SCT. There has been one NRM (cGVHD) and one relapse mortality. The OS at one year is 66.7%. Conclusions: MA AutoSCT followed by RI AlloSCT is feasible and well tolerated in pediatric pts with R/R HD. A larger study with longer follow up is required to determine if this approach will reduce relapse, long term toxicity and/or improve survival. No significant financial relationships to disclose.

Unpaired Median Neurones in a Lepidopteran Larva (Antheraea Pernyi): II. Peripheral Effects and Pharmacology

Two unpaired median cells (MC1 and MC2) had a temporal pattern of firing that correlated with phasic muscular activity in preparations of larval Antheraea pernyi, and previous work has indicated that the axons of median cells are associated with nerve trunks innervating blocks of muscle. In spite of this, action potentials in median cells were not found to have any one-for-one effects on either the tension or the electrical activity of somatic muscle fibres. However, bursts of action potentials in MC2 were shown to modulate both tension production and electrophysiological activity of a number of motor units. These effects consisted of an increase in twitch tension, a relaxation of basal resting tension, an increase in relaxation rate following contractions, a hyperpolarization of some muscle fibres and an increase in amplitude of excitatory junction potentials. The relative potency of these different effects varied between fast and slow muscles. All of these effects were mimicked by the application of octopamine and synephrine, and in higher concentrations by a number of other biogenic amines and adrenergic agonists. The possibility that the effects of median cell activity were mediated by the release of endogenous octopamine was supported by the observation that phentolamine (10−5mol l−1) blocked the effects of both MC2 impulses and the application of exogenous octopamine, whereas propanolol affected neither set of responses. This observation also indicated a pharmacological similarity with a number of other octopamine-sensitive insect tissue preparations. MC1 had similar effects to MC2 on the electrical activity of a number of muscles, suggesting that these two cells play a similar role. These observations provide strong evidence for the presence of an identifiable octopaminergic system of neurones, similar to the dorsal unpaired median (DUM) neurones that have been extensively studied in the locust.