Daily Cell Counts Research Articles

A single intranasal dose of essential oil spray confers modulation of the nasopharyngeal microbiota and short-term inhibition of Mannheimia in feedlot cattle: a pilot study

Five essential oils (EOs) were previously characterized in vitro and identified as candidate EOs for the development of an intranasal EO spray to mitigate bovine respiratory disease (BRD) pathogens. In the present study, these EOs were evaluated for their potential to (i) reduce BRD pathogens, (ii) modulate nasopharyngeal microbiota, and (iii) influence animal performance, feeding behavior and immune response when a single dose administered intranasally to feedlot cattle. Forty beef steer calves (7–8 months old, Initial body weight = 284 ± 5 kg [SE]) received either an intranasal EO spray (ajowan, thyme, fennel, cinnamon leaf, and citronella) or PBS (Control; n = 20/group) on day 0. Deep nasopharyngeal swabs were collected on days (d) -1, 1, 2, 7, 14, 28, and 42 and processed for 16S rRNA gene sequencing, qPCR, and culturing. Significant effects of EO on community structure (d1), microbial richness and diversity, relative abundance of some dominant phyla (d1, d2, and d14), and the overall interaction network structure of the nasopharyngeal microbiota were detected. The relative abundance of Mannheimia was lower in the EO calves (4.34%) than in Control calves (10.4%) on d2, and M. haemolytica prevalence on d7 as compared to control calves. Feed intake, average daily gain, feeding behavior, and blood cell counts were not affected by EO treatment. Overall, a single intranasal dose of EO spray resulted in moderate modulation of nasopharyngeal microbiota and short-term inhibition of Mannheimia while not influencing animal performance, feeding behavior or immune response. Our study, for the first time, shows the potential use of intranasal EO to mitigate BRD in feedlot cattle.

Abstract 3787: miR 2355-5p regulates tumor growth and angiogenesis in VHL-inactivated clear cell renal cell carcinoma

Abstract Background: Clear Cell Renal Cell Carcinoma (ccRCC) constitutes more than 75% of kidney cancer diagnostics. Inactivation of the tumor suppressor gene termed von Hippel-Lindau (VHL) is considered the major truncal event and occurs in more than 85% of patients. The most notable role of VHL is its ability to negatively regulate the Hypoxia-Induced Factors (HIFα), which in turn, stimulate the transcription of multiple oncogenes, including microRNAs (miRNAs). MiRNAs are small non-coding RNAs that negatively regulate gene expression through complementary base pairing. Using ccRCC cell models, we observed an overexpression of the miR 2355-5p following the loss of VHL in a HIF2α dependent manner. Hypothesis: We hypothesize that the miR 2355-5p overexpression is involved in the tumorigenesis of VHL-inactivated ccRCC. Our specific aims are to i) quantify the expression of miR 2355-5p in ccRCC clinical samples, ii) characterize the functional impact of miR 2355-5p in ccRCC development, and iii) identify miR 2355-5p targeted genes in ccRCC tumors. Methods: Clinical samples (tissues and plasma) from consented patients with ccRCC were recruited through the Université de Sherbrooke. Normal adjacent tissue and plasma from healthy individuals were used as controls. miR 2355-5p expression was quantified through RT-qPCR with Taqman probes. Then, 786-0 and A498 VHL-deficient ccRCC cell lines were used. miR 2355-5p knockout models (KO) were generated using CRISPR/cas9 technology or stably overexpressed using miRNA precursor plasmid (786-0++2355). The impact on cell survival and proliferation was assessed with clonogenic assay and daily cell counts, respectively. Furthermore, cell models were injected into immunodeficient mice. Angiogenesis was evaluated by tube formation assay and angiogenesis proteome profiler kit. Finally, miRNA pulldown was performed to identify miR 2355-5p potential targets. Results: Our findings showed a significant increase in miR 2355-5p expression in tumor tissue compared to normal adjacent tissue as well as in plasma samples compared to healthy individuals. Inhibition of miR 2355-5p significantly reduced cell survival and cell proliferation in vitro and in vivo. Altered expression of the miRNA also disturbed the expression of multiple angiogenic factors and reduced the ability of cells to stimulate tube formation. MiRNA pulldown identified over 267 potential targets, where 161 were mRNA coding for functioning proteins. Close to 80% of identified proteins were direct targets of miR 2355-5p according to prediction software. Further analysis showed an inverse correlation between the tumor suppressor gene SPRY4 and miR 2355-5p, uncovering a potential link between the two. Discussion: Our results demonstrate an important role of miR 2355-5p on angiogenesis and ccRCC tumor growth. Further research on the interaction between miR-2355-5p, BTG2, SLIT2, and CMTM4 could unlock new potential avenues for targeted therapies. Citation Format: Patric Page, Sandra Turcotte, Mykella Martin, Patrick Richard, Tamiko Nishimura, Yasser Riazalhosseini. miR 2355-5p regulates tumor growth and angiogenesis in VHL-inactivated clear cell renal cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3787.

Evaluation of the influence of different culture media on growth, life cycle, biochemical composition, and astaxanthin production in Haematococcus pluvialis

Haematococcus pluvialis is known as the richest natural source of carotenoid astaxanthin, whose synthesis is related to changes in cells according its complex life cycle and the availability of nutrients present in the culture media. This study’s objective was to evaluate the influence of different culture media under photoautotrophic and mixotrophic conditions on the growth, life cycle, biochemical composition, and astaxanthin production in H. pluvialis. Cultures were performed using the following media: KM1, MM2, Provasoli, modified Provasoli, and how to control the BBM, all kept at 24 ± 1 °C, with constant aeration, light/dark photoperiod 24:0 and irradiance of 60 μmol photons/m2/s. Growth and life cycle were evaluated by daily cell counts under an optical microscope; values of maximum cell density (MCD) and dry biomass were also assessed. Biochemical analyses were chlorophyll (a and b), astaxanthin, total proteins, and fatty acids. Cultivation under photoautotrophic conditions in BBM medium showed higher concentration of chlorophylls a (33 ± 4.0 μg/mL) and b (18 ± 1.6 μg/mL) and protein (62.7 ± 3.38%). Cultivation in modified Provasoli medium, under mixotrophic conditions, showed higher MCD (84.5 ± 11.7), biomass production (4.9 ± 0.0004 mg/mL), fatty acid methyl esters, and astaxanthin productivity (9.28 ± 0.4 mg/L/day). These results demonstrate that the culture media and the culture conditions to which H. pluvialis is submitted stimulate specific metabolic and biosynthetic pathways of the cells, which directly affect the biochemical composition of the microalgae. Modified Provasoli medium proved to be the most efficient in increasing astaxanthin productivity for this species, as it has shown better growth parameters and biomass production.

CRISPR Mediated ELANE Single-Allele Knock-out Restores Proliferation and Myeloid Differentiation of Neutropenia Patient Derived BM HSCs

Background: Mutations in ELANE are a cause of cyclic and severe congenital neutropenia (SCN), predominantly autosomal dominant disorders. ELANE encodes neutrophil elastase (NE), a tissue specific serine protease. Neutropenia occurs because mutant NE impairs survival and maturation of myeloid cells. More than 130 disease causing mutations in ELANE have been identified and cell permeable inhibitors of NE can correct the defect in cell survival and maturation in cellular models. (Makaryan et al. J Leukoc Biol. 2017;102:1143). CRISPR/Cas9 knock-out (KO) of ELANE in patients' hematopoietic stem cells (HSCs) corrects myeloid cell differentiation, (Nasri et al Haematologica 2020:105:598), but this approach targets both alleles, eliminates normal NE and may impair innate immunity. Hypothesis: Selective, single allele editing is a preferred strategy for correcting ELANE associated neutropenia and other autosomal dominant disorders. Methods: Emendo Biotherapeutics developed CRISPR-associated allele specific KO based on single nucleotide polymorphisms (SNPs) located in the vicinity of ELANE. Three SNPs cover ~80% of the population. In the current study, editing of the mutated allele was achieved by targeting SNP rs1683564 located downstream to the 3'UTR of ELANE and mediating a biallelic break in intron 4, with excision of the 3'UTR, destabilized the mutated allele transcript. For editing we utilized RNPs assembled from Emendo's proprietary nuclease, OMNI-50, a guide targeting the SNP and a guide targeting a region in intron 4. OMNI-50 has low off-target activity and high allele specific editing when targeting either the reference or the alternative form of the SNPs. The efficacy of this composition was tested on normal and SCN patient-derived HSCs harboring the relevant SNP. (Figure 1) A patient's bone marrow CD34+ cells with the S126L mutation and rs1683564 were enriched using RosetteSep™ Progenitor Cell Enrichment Cocktail (Stemcell Technologies), purified by density gradient centrifugation using Lymphoprep (Stemcell Technologies). The purified CD34+ cells were expanded 4 days in StemSpan™ SFEM II media, supplemented with StemSpan™ CD34+ Expansion Supplement. Expanded CD34+ cells were further purified using StemSep™ Human CD34 Positive Cocktail and purity verified by FACS analysis using CD34 and CD45 antibodies. Purified CD34+ cells were cryopreserved using serum free CryoStor® CS10 media (Stemcell Technologies) and stored in Liquid Nitrogen Vapor Phase. Normal human blood CD34+ progenitor cells, harboring rs1683564 were similarly treated as the control. Allele specific excision was determined by ddPCR. To determine excision efficiency for the patient and control, we amplified two regions in ELANE, Exon 1 and Exon 5 (excised region), using two different probes labeled with FAM and HEX, respectively. We used OMNI-50 nuclease (Emendo Bio, Ness Ziona, Israel), electroporation (Lonza 4D nucleofector) and StemCell media (Stemcell Technologies, Vancouver, BC) to modify, grow and differentiate the CD34+ cells. We assessed myeloid cell proliferation and differentiation using daily cell counts, cytospins stained with Diff-Quik, and CD14, CD66b, CD15, CD16, CD11b labeling and flow cytometry. Results: Unedited patient cells demonstrated significant abnormalities in proliferation and differentiation. Cytospins from unedited patient CD34+ cells at 14 days showed block of myeloid differentiation and 4-fold greater monocytes compared to control, consistent with the hematopoietic defect in SCN patients. Single allele ELANE KO significantly improved these cellular abnormalities: total cell proliferation increased 41% and CD14/CD66b and CD15/CD11b positive cells increased by 107% and 65.5%, respectively. Studies are in progress for additional patients harboring different ELANE mutations. Conclusions: Single allele KO may resolve concerns about unwanted effects of total elimination of the normal gene products with gene editing for autosomal dominant diseases and be an effective strategy for treating ELANE associate neutropenia. Disclosures Sabo: Emendo BioTherapeutics: Research Funding. Makaryan:Emendo BioTherapeutics: Research Funding. Poulsen:Emendo BioTherapeutics: Research Funding. Povodovski:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Dicken:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Herman:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Emmanuel:Emendo BioTherapeutics: Current Employment, Current equity holder in private company. Dale:X4 Pharmaceuticals: Research Funding; Emendo BioTherapeutics: Consultancy; X4 Pharmaceuticals: Honoraria.

Expansion of umbilical cord blood derived haematopoietic stem cells under constant dynamic conditions

Background & Aim Umbilical cord blood (UCB) offers a source of haematopoietic stem and progenitor cells (HSPCs) with a proven clinical rack record. However, one major drawback is the relatively low number of HSPCs within one unit, limiting its clinical potential. Due to this, strategies to expand UCB-HSPCs have been developed and involve the use of additional molecules to support expansion, such as SR-1, UM-171, immobilised Notch ligand Delta-like 1 and nicotinamide. As these potential therapeutics reach late stage clinical trials, considerations for manufacturing in order to meet clinical demand are required. In this study we have investigated the impact that constant shaking, at differing magnitudes, during the expansion of UBC-HSPCs has on the overall total nucleated cell expansion, Lin-CD34+ and Lin-CD34+CD38-CD90+ cell expansion, cell viability and colony forming unit (CFU) potential. Methods, Results & Conclusion Method UCB CD34+ cells were seeded at 1 × 104 cells/mL in IMDM+10% FBS supplemented with 100ng/mL SCF, TPO and Flt3-L and 20ng/mL IL-6 and maintained under static (T-flasks) or shaken (125mL Erlenmeyer flasks, with a 10mm shaking diameter) conditions for nine days. Daily cell counts were completed and immunophenotyping, apoptosis detection and CFU potential were performed on day 0 and day 9. Results UCB-HSPCs were successfully expanded for all donors under both conditions, the final viable cell densities achieved were significantly higher in the shaken conditions compared to the static controls, in addition the final viable cell densities increased with an increasing agitation rate, 3.1±0.3 × 104, 6.2±0.1 × 104, 10.2±0.2 × 104 and 12.4±0.4 × 104 cells mL−1 (mean value ± SD) for static, 30rpm, 70rpm and 110rpm respectively. Immunophenotyping of the expanded cells demonstrated an increase in both Lin-CD34+ HSPCs and Lin-CD34+CD38-CD90+ haematopoietic stem cells, this increase was significantly higher in the shaken conditions compared to the static controls. Finally, the fold change in CFU potential was significantly higher for both granulocyte, monocyte colonies and granulocyte, erythrocyte, monocyte, megakaryocyte colonies compared to static controls and with an increasing agitation rate. Conclusion In this study we have shown that expansion of UCB-HSPCs under shaken conditions improves the overall cellular yield that can be achieved, maintains both Lin-CD34+ and the more primitive Lin-CD34+CD38-CD90+ cell populations and enhances CFU potential.

English

Tea Tree oil (TTO), the essential oil from the Australian native Melaleuca alternifolia has demonstrated a variety of beneficial efficacies including antimicrobial, antifungal, antiviral and anti-inflammatory. This report discusses data obtained on the in vitro activity of TTO on human glioblastoma cells U87MG. Cell viability was examined by 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide MTT assay. Growth was investigated by incubating cells with various concentrations of TTO (0.025 and 0.05 %) for 24, 48 or 72 h and daily cell count. Cell cycle and apoptosis assay were assessed by flow citometry. TTO decreased cell viability in a dose and time-dependent manner. . The cell cycle distribution showed that TTO enhanced the accumulation of the cells in G0/G1 phase. The analysis by Western blot of protein related to cell cycle (CDK2 and p27), cell apoptosis (caspase 6 and 8), necrosis (TNFR1 and RIP1) demonstrated that TTO induces U87MG growth inhibition by more synergic mechanisms: necrosis, low level apoptosis and cell cycle arrest. TTO induces also in vivo glioblastoma tumor growth inhibition in a murine subcutaneous model. Key words: Brain cancer, tea tree oil, terpinol, glioblastoma, natural drug, adjuvant chemotherapy, temozolomide, apoptosis, cell cycle.    

Effect of altered nitrogen partial pressure on Chlorellaceae for spaceflight applications

Algal-based photobioreactors offer a promising bioregenerative technology that can perform the functions of air revitalization, water recycling, and food production in a human spacecraft life support system. In order to assess their performance in this context, this study exposed a non-axenic green algal culture (Chlorellacea) to an atmospheric pressure of 8.2psia [56.5 kPa] with an oxygen concentration of 34%, which are currently proposed cabin atmosphere conditions for future space exploration missions. Growth and metabolism of green algae were compared to a control experiment kept at 14.7psia [101.3 kPa] with oxygen and carbon dioxide partial pressure maintained at constant levels of 3.1 and 0.147psia [21.3 and 1.0 kPa], respectively, which represent concentrations of 21 and 1% at standard sea level pressure. To conduct the evaluation, a flow-through photobioreactor was developed that can precisely control atmospheric compositions such as partial pressures of oxygen and carbon dioxide and set the total pressure solely by altering the nitrogen partial pressure. Algal performance was measured by daily cell counts and optical density measurements. Additionally, continuous measurements of dissolved oxygen and pH in the liquid phase, as well as evolved oxygen and absorbed carbon dioxide in the gaseous phase, were collected. A metagenomic analysis was conducted on the algal culture to assure the composition of the algal culture remained consistent during the experimental phase, as well as to characterize the nature of coexisting contaminants in the non-axenic culture. The performance of the algal culture was compared to related, previously published literature values. The hypothesized increase in performance of the algal culture under reduced pressures was not verified, rather it was shown that the nitrogen partial pressure neither benefits nor inhibits algal growth under the tested conditions.

Economic selection index in small rural dairy farms.

This study aimed to calculate economic values (EVs) and economic selection indices for milk production systems in small rural properties. The traits 305-d milk yield in kg (MY), fat (FP) and protein (PP) percentage, daily fat (FY) and protein (PY) yield, cow live weight in kg (LW), calving interval (CI), and logarithm of daily somatic cell count (SCC) in milk were considered the goals and selection criteria. The production systems were identified from 29 commercial properties based on the inventory of revenues and costs and of zootechnical field data. Later, bioeconomic models were developed to calculate the productive performance, revenues, and costs concerning milk production to estimate EVs, which were calculated as the difference in annual profit with dairy production resulting from a change in one unit of the trait while keeping the others constant and dividing the value by the number of cows. After the EVs were known, ten economic selection indices were estimated for each system so they could be compared by modifying the selection criteria and calculating the relative importance of each selection criteria, the accuracy of the economic selection index, and response expected to the selection in USD, among other parameters. One of the systems detected was called less intensive (LS) and was characterized by having ten cows in lactation that produced 13·5 l/d and consumed 1·8 kg of concentrate/d. The second system detected was called more intensive (IS) and had 22 cows in lactation that produced 17·5 l/d and consumed 3·4 kg of concentrate/d. Monthly profits per cows in lactation of USD 2·60 and USD 68·77 were recorded for LS and IS, respectively. The EVs of the traits MY, FP, and PP were all positive, while for the other traits they were all negative in all situations. The best economic selection indices were those featuring selection criteria MY, LW, and CI, while the trait LW had the greatest importance in both systems. These results indicate that animal frame must be controlled in order to maximize the system's profit.

Personalized and Optimized Low-Dose and Intensive Chemotherapy Treatments for Patients with Acute Myeloid Leukemia (AML)

Patients with AML have heterogeneous features, including those specific to the patient as well as those specific to the disease, such as leukemic burden and dynamic sub-clonal populations. Outside of clinical trials, few of these components are used to determine treatment. In order to move towards precision medicine, we have developed πiChemo, a computational application based on a dynamic mathematical modelling framework, using patient-, leukemia- and treatment-specific data to predict outcomes and optimize chemotherapy regimens for patients with AML.The model consists of a pharmacokinetic and pharmacodynamic (PK/PD) module that calculates the concentration and effect of Cytarabine Arabinoside (Ara-C) and Daunorubicin (DNR) in bone marrow (BM); and a population balance models (PBMs) module that describes normal populations (stem cells, progenitors, precursors) and abnormal populations (leukemic sensitive blasts (LSB) and leukemic resistant blasts (LRB)) in BM. The PBMs module also determines mature cell numbers in three lineages found in BM and peripheral blood (PB): (1) red blood cells (RBC), (2) white blood cells (WBC) and (3) lymphocytes (L). Model structure was analysed by global sensitivity analysis, which identified the most significant parameters on outcome predictions, re-estimated for each patient. The final integrated PK/PD & PBMs model has 1,295 differential equations, 8,044 algebraic equations, 9,335 variables, 25 fixed parameters and 4 degrees of freedom or variables to be optimized (Ara-C dose, Ara-C injection duration, DNR dose and DNR injection duration).Model validation, predictions and optimizations were performed using anonymised retrospective data from 28 patients with AML. The model required: (i) patient features: height, weight, age and gender, (ii) patient status: initial BM differential and PB cell counts, (iii) leukemia data: cellularity, presence of dysplasia and initial blast percentage and, (iv) treatment data: type (low-dose (LD) or intensive (DA)), dose, administration route (SC vs IV), administration mode (bolus injection vs infusion), time between injections and between cycles.The model predicted the absolute numbers of stem cells, progenitors, precursors, WBC, RBC, L, LSB and LRB in BM, and WBC, RBC, L and neutrophil count in PB during treatment for all patients. Model simulations predicted outcomes for 18 patients who achieved complete remission (7 LD & 11 DA), 4 patients who entered partial remission (2 LD & 2 DA) and 6 patients who relapsed (2 LD & 4 DA). The most remarkable results are those of prediction for BM blast percentage after each chemotherapy cycle and the PB neutrophil count for all patients. The notable fit between model predictions and daily patient data demonstrate model robustness and accuracy in the capacity to track patient-specific restaging BM and daily PB count evolution before, during and after treatment.The same patient datasets were used to apply an optimization algorithm that could maximize normal cell number and reduce leukemia burden, to personalize chemotherapy dose and administration for best outcomes. The results show that doses and administration methods vary between patients and between chemotherapy cycles for the same patient, depending on the evolution of normal and abnormal populations in BM. Low-dose continuous Ara-C infusions were more effective than rapid bolus injections, due to reduced chemotherapy effects on normal cells and subsequent quicker recovery in the normal BM compartments. RBC progenitors and precursors recovered faster than WBC and L lineages, and the recovery of normal BM cells was faster than that of normal mature cells in PB.The πiChemo tool requires only patient- and leukemia-specific initial conditions at diagnosis, easily obtained in standard clinical practice, for outcome predictions and treatment optimizations. Real-time model-fit testing and comparison of model results against daily PB cell counts would enable the re-estimation of significant parameters, increasing model accuracy and treatment effectiveness whilst therapy is ongoing. The πiChemo precision therapy tool has the potential to personalize optimal standard and novel treatments for AML in real-time. DisclosuresNo relevant conflicts of interest to declare.

Effects of aloe emodin on U87MG glioblastoma cell growth: In vitro and in vivo study.

Glioblastoma, the most aggressive and malignant form of glioma, appears to be resistant to various chemotherapeutic agents. Hence other approaches have been investigated to target more pathways involved in glioblastoma development and progression. Here we investigate the anticancer effect of Aloe-Emodin (AE), an anthraquinone compound presents in the leaves of Aloe arborescens, on human glioblastoma cell line U87MG. U87MG were treated with various concentrations of AE (20 and 40 μM) for different times (24, 48, and 72 hr). Cell growth was monitored by daily cell count after treatments. Growth analysis showed that AE significantly decrease proliferation of U87MG in a time and dose dependent manner. FACS analysis demonstrates a block of cell cycle in S and G2/M phase. AE probably induced also apoptosis by releasing of apoptosis-inducing factor: PARP and Lamin activation leading to nuclear shrinkage. In addition, exposure of U87MG to AE reduced pAKT phosphorylation. AE inhibition of U87MG growth is a result of more mechanism together. Here we report that AE has a specific growth inhibition on U87MG also in in vivo. The growth of U87MG, subcutaneously injected in nude mice with severe combined immunodeficiency, is inhibited without any appreciable toxic effects on the animals after AE treatment. AE might represent a conceptually new lead antitumor adjuvant drug.

Effects of hispolon on glioblastoma cell growth.

Hispolon is a polyphenolic compound isolated from Phellinus linteus which exhibits antitumor activity. Here, we explored the effects of hispolon on human glioblastoma cells U87MG. Cell viability was examined by MTT assay. Growth was investigated by incubating cells with various concentrations of hispolon (25 and 50 µM) for 24, 48 or 72 h and daily cell count. Cell cycle and apoptosis assay were assessed by flow cytometry. Hispolon decreased cell viability in a dose- and time-dependent manner. The cell cycle distribution showed that hispolon enhanced the accumulation of the cells in G2/M phase. Hispolon decreased the expression of G1-S transition-related protein cyclin D4 but increased the expression of CDK inhibitor p21. Additionally, hispolon enhanced the expression of p53. Moreover, hispolon treatment was effective on U87MG cells in inhibiting cell viability and inducing cell apoptosis. Our results indicate that hispolon inhibits the cell viability, induces G2/M cell cycle arrest and apoptosis in glioblastoma U87MG cells, and p53 should play a role in hispolon-mediated antitumor activity.

Progesterone and Melanoma Cells: An Old Story Suspended between Life and Death

Melanoma is a widespread cancer with poor prognosis. Female hormones are known to be capable of influencing melanoma progression but clinical data related to pregnancy, oral contraception and hormone replacement therapy are controversial. A few reports show that in vitro progesterone (PG) affects melanoma growth in nuclear progesterone receptor (nPR)-positive and nPR-negative cells, but the experimental protocols used are quite different and the results are not univocal. Further research on this topic is thus needed especially in view of the widespread use of PG in clinical practice. In this study, we used human melanoma cells (A-375), which were cultured in vitro in the presence or absence of a wide range of PG concentrations (from 0.01 to 1000 M) in single treatment. Daily cell count, cell cycle analysis and apoptosis assay were performed. Our results show that the low PG concentrations (from 0.01 to 1.0 M) promote a significant increase of melanoma cell proliferation but this growth-stimulatory effect is not observed at 10 M PG and the higher PG concentrations (i.e. 100 and 1000 M) induce a cell density reduction which is the result of both cell cycle arrest and apoptosis. These findings confirm and extend previous observations reported in the international literature. A higher caution in the clinical use of progesterone is thus mandatory, since PG concentrations capable of stimulating melanoma cell proliferation are very close to those commonly used in a wide spectrum of physio-pathological conditions.

The proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°Celsius compared to euthermic conditions in pigs

Given similarities in metabolic parameters and cardiovascular physiology, the pig is well positioned as a biomedical model for metabolic disease and obesity in humans. Better understanding molecular mechanisms governing porcine adipocyte hyperplasia may provide insight into the regulation of adipose tissue development that is useful both when considering the pig as a commodity and when extrapolating porcine data to human disease. Primary cultures of pig stromal-vascular cells have served as a useful tool for investigating factors that regulate preadipocyte proliferation and differentiation. However, such cultures have generally been maintained at 37°C in vitro despite euthermia being 39°C in pigs. To address potential concerns about the physiological relevance of culturing primary pig preadipocytes under what would be hypothermic conditions in vivo, the objective of this study was to investigate the effect of culture temperature on the proliferation and differentiation of pig preadipocytes in primary culture. Culturing primary preadipocytes at 37 rather than 39°C decreases their proliferation rates based upon cleavage of the tetrazolium salt, MTT (P < 0.001), reduction of resazurin (P < 0.001), and daily cell counts (P < 0.001). Likewise, culturing primary porcine preadipocytes at 37°C suppressed their adipogenic potential based upon monitoring adipogenesis morphologically, biochemically, and via the expression of mRNA encoding adipogenic marker genes. Collectively, these data indicate the proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°C compared to normal body temperature of pigs. This may confound investigation of factors that impact adipocyte hyperplasia in the pig.

An assessment of the usefulness of the cyanobacterium Synechococcus subsalsus as a source of biomass for biofuel production

Nowadays algal biofuels are considered one of the most promising solutions of global energy crisis and climate change for the years to come. By manipulation of the culture conditions, many algal species can be induced to accumulate high concentrations of particular biomolecules and can be directed to the desired output for each fuel. In this context, the present study involved the assessment of the effects of CO 2 availability and nitrogen starvation on growth and chemical composition of the cyanobacterium Synechococcus subsalsus , testing a fast-growing native strain. The control experiments were performed with Conway culture medium in 12-day batch cultures, in 6-liter flasks and 12 h photoperiod, with addition of 2 L min -1 filtered air to each flask. Other two experimental conditions were also tested: (i) the placement into the cultures of additional dissolved nutrients except nitrogen, one week after the start of growth (N-), and (ii) the input of pure CO 2 into the flasks from the 5th day of growth (C+). In all cultures, daily cell counts were done throughout the cultivation, as well as measurements of pH and cell biovolumes. Maximum cell yield were found in N-experiments, while cell yields of C+ and control were similar. Dissolved nitrogen was exhausted before the end of the experiments, but dissolved phosphorus was not totally consumed. Protein and chlorophyll-a concentrations decreased from the exponential to the stationary growth phase of all experiments, except for protein in the control. In all experiments, carbohydrate, lipid and total carotenoid increased from the exponential to the stationary growth phase, as an effect of nitrogen limitation. Increments in carbohydrate concentrations were remarkable, achieving more than 42% of the dry weight (dw), but concentrations of lipid were always lower than 13% dw. The addition of pure CO 2 did not cause a significant increase in biomass of S. subsalsus nor generated more lipid and carbohydrate than the other treatments. Nitrogen starvation caused an intense accumulation of carbohydrate, but the increments of lipid were small. Despite the fast growth, the cyanobacterium S. subsalsus has a virtually null potential for biodiesel production, given its low lipid concentrations. The high concentrations of carbohydrate combined with fast growth point to the potential use of this species as raw material for other possible biotechnological processes, after a demonstration of technical and economic viability.

Effect of estrus synchronization on daily somatic cell count variation in goats according to lactation number and udder health status

Two repeated experiments were carried out in 2 different years to study the effect of estrus on somatic cell count (SCC) in dairy goats. In the first year, 36 Murciano-Granadina goats were used [12 primiparous and 24 multiparous; 22 healthy and 14 with an intramammary infection (IMI)] and, after a 6-d pre-experimental period, were divided into 2 groups according to lactation number, udder health status, SCC, and milk production. One group was kept as a control, whereas the other received an estrus synchronization hormonal treatment lasting 11d. At 24, 48, and 72h after cessation of the hormone treatment, goats were placed in contact with a buck to confirm that they were in estrus. For 32 consecutive days (6 pre-experimental, 11 in hormone treatment, and 15 post-treatment) the SCC per gland and udder were monitored in all animals. In the second year, we repeated the same experimental design using a total of 38 Murciano-Granadina breed goats (12 primiparous and 26 multiparous; 26 healthy and 12 with IMI). Throughout this experiment, milk yield and composition were also recorded daily for each goat. Upon termination of the hormonal treatment, the SCC in udder milk increased significantly in the treatment group compared with the control group over 3 consecutive days. This increase was observed for year (1 and 2), parity (primiparous and multiparous), and udder health status (healthy and IMI). The log10 SCC (cells/mL) increased from 5.5±0.09 before estrus to 6.04±0.09 during treatment; therefore, the geometric mean of the SCC increased 3.5 times during treatment. The maximum values obtained in healthy glands of primiparous goats (geometric mean=0.37 million cells/mL) were lower than in healthy glands (1.1 million cells/mL) or infected glands (1.7 million cells/mL) of multiparous goats. The increase in SCC observed during estrus (200% increase in geometric means) could not be explained by the changes in milk production, which only fell by 13%. During estrus, the percentage of protein and dry matter in the milk also increased significantly. We concluded that it is necessary to consider the presence of estrus to correctly interpret milk SCC, as an indirect method for detecting IMI or as a commercial milk quality parameter.

Characterization of human vocal fold fibroblasts derived from chronic scar

In vitro modeling of cell-matrix interactions that occur during human vocal fold scarring is uncommon, as primary human vocal fold scar fibroblast cell lines are difficult to acquire. The purpose of this study was to characterize morphologic features, growth kinetics, contractile properties, α-smooth muscle actin (α-SMA) protein expression and gene expression profile of human vocal fold fibroblasts derived from scar (sVFF) relative to normal vocal fold fibroblasts (nVFF). In vitro. We successfully cultured human vocal fold fibroblasts from tissue explants of scarred vocal folds from a 56-year-old female and compared these to normal fibroblasts from a 59-year-old female. Growth and proliferation were assessed by daily cell counts, and morphology was compared at 60% confluence for 5 days. Gel contraction assays were evaluated after seeding cells within a collagen matrix. α-SMA was measured using western blotting and immunocytochemistry (ICC). Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was used to assess differential extracellular matrix gene expression between the two cell types. sVFF were morphologically indistinguishable from nVFF. sVFF maintained significantly lower proliferation rates relative to nVFF on days 3 to 6 (day 3: P = .0138; days 4, 5, and 6: P < .0001). There were no significant differences in contractile properties between the two cell types at any time point (0 hours: P = .70, 24 hours: P = .79, 48 hours: P = .58). ICC and western blot analyses revealed increased expression of α-SMA in sVFF as compared with nVFF at passages 4 and 5, but not at passage 6 (passage 4: P = .006, passage 5: P = .0015, passage 6: P = .8860). Analysis of 84 extracellular matrix genes using qRT-PCR revealed differential expression of 15 genes (P < .01). nVFF and sVFF displayed differences in proliferation rates, α-SMA expression, and gene expression, whereas no differences were observed in contractile properties or morphology. Further investigation with a larger sample size is necessary to confirm these findings.

Inhibition of STAT-3 results in greater cetuximab sensitivity in head and neck squamous cell carcinoma

ObjectiveThe inhibition of epidermal growth factor receptor (EGFr) with the monoclonal antibody cetuximab reduces cell proliferation and survival which correlates with increased DNA damage. Since the signal transducer and activator of transcription-3 (STAT-3) is involved in the EGFr-induced signaling pathway, we hypothesized that depletion of STAT-3 may augment cetuximab-induced processes in human head and neck cancer cells. Materials and methodsHuman head and neck squamous carcinoma cells (UM-SCC-5) were transfected with short hairpin RNA (shRNA) against STAT-3 (STAT3-2.4 and 2.9 cells). A mutated form of this shRNA was transfected for a control (NEG4.17 cells). Radiosensitivity was assessed by a standard colony formation assay. Proliferation was assessed by daily cell counts following treatment and apoptosis was assessed by an annexin V-FITC assay. The alkaline comet assay was used to assess DNA damage. ResultsThe STAT-3 knockdown cells (STAT3-2.4 and STAT3-2.9 cells) demonstrated enhanced radiosensitivity compared to control NEG4.17 cells, which correlated with increased apoptosis. Also, the STAT-3 knockdown cells demonstrated decreased proliferation with cetuximab treatments compared to control cells (NEG4.17). The increased cetuximab sensitivity of the STAT-3 knockdown cells correlated with increased apoptosis and DNA damage compared to control cells (NEG4.17). ConclusionThese studies revealed that the greater anti-proliferative effects and increased cytotoxicity of cetuximab in the STAT3-2.4 and STAT3-2.9 cells compared to control NEG4.17 cells, may be a result of STAT3-mediated effects on cellular apoptosis and DNA damage.

IL-21 Contributes to JAK3/STAT3 Activation and Promotes Cell Growth in ALK-Positive Anaplastic Large Cell Lymphoma

Interleukin (IL)-21 has been reported to both stimulate cell growth and promote survival in benign lymphoid cells and several types of hematopoietic neoplasms. It induces JAK3/STAT3 signaling, a biologically important cellular pathway activated in most cases of anaplastic lymphoma kinase (ALK)-expressing anaplastic large cell lymphoma (ALK(+)ALCL). Therefore, we hypothesize that IL-21 may contribute to JAK3/STAT3 activation and cell growth in ALK(+)ALCL. By reverse transcription-PCR, we found consistent expression of IL-21 receptor (IL-21R) in all ALK(+)ALCL cell lines and frozen tumors examined. IL-21 was also consistently expressed in ALK(+)ALCL tumors, although its mRNA was detectable in only one of three cell lines tested. By immunohistochemistry, we examined 10 paraffin-embedded ALK(+)ALCL tumors; all cases were positive for both IL-21 and IL-21R in these neoplastic cells. IL-21 signaling is biologically significant in ALK(+)ALCL since the addition of recombinant IL-21 enhanced the activation of JAK3/STAT3 and significantly increased cell growth in ALK(+)ALCL cell lines. However, small interfering RNA down-regulation of IL-21R significantly decreased both STAT3 activation and cell growth. IL-21R expression is not linked to nucleophosmin-ALK since forced expression of nucleophosmin-ALK and small interfering RNA down-regulation of nucleophosmin-ALK did not significantly change the expression of either IL-21R or IL-21. Our findings thus support the enhancement of JAK3/STAT3 activation and cell growth in ALK(+)ALCL via IL-21 signaling. These results further support the concept that constitutive activation of STAT3 in these tumors is multifactorial.

An Effective and Less Invasive Treatment of Post-Traumatic Cerebrospinal Fluid Fistula: Closed Lumbar Drainage System

Cerebrospinal fluid (CSF) leakage may develop when a defect is formed in dural layers by traumatic or iatrogenic processes. Traumatic CSF leakage was encountered in 2% of head injuries. CSF leakage is an associated feature of 12-30% of skull base fractures. Numerous treatment modalities are proposed for the management of CSF leaks. A closed lumbar drainage system (CLDS) is recommended as an alternative method to surgery for preventing complications related to leakage. In the present study, we report the clinical details of 46 patients who suffered from rhinorrhea/otorrhea (R/O), three of whom were urgently operated due to other causes like subdural hematoma and tension pneumocephalus. Leakage spontaneously ceased in 26 (60.4%) patients whereas CLDS insertion was found to be necessary in 17 patients. A prophylactic antibiotic regimen was started in the CLDS group and these patients were followed with daily CSF cell counts. Fifteen patients (88.2%) were successfully treated by CLDS, however meningitis developed in 2 patients (11.7%). Severe complications like meningitis could be avoided by a simple daily microbiological analysis of CSF.

Neonatal stem cells exhibit specific characteristics in function, proliferation, and cellular signaling that distinguish them from their adult counterparts

Stem cells may be a novel treatment modality for organ ischemia, possibly through beneficial paracrine mechanisms. Stem cells from older hosts have been shown to exhibit decreased function during stress. We therefore hypothesized that 1) neonatal bone marrow mesenchymal stem cells (nBMSCs) would produce different levels of IL-6, VEGF, and IGF-1 compared with adults (aBMSCs) when stimulated with TNF or LPS; 2) differences in cytokines would be due to distinct cellular characteristics, such as proliferation or pluripotent potential; and 3) differences in cytokines would be associated with differences in p38 MAPK and ERK signaling within nBMSCs. BMSCs were isolated from adult and neonatal mice. Cells were exposed to TNF or LPS with or without p38 or ERK inhibition. Growth factors were measured via ELISA, proliferation via daily cell counts, cell surface markers via flow cytometry, and pluripotent potential via alkaline phosphatase activity. nBMSCs produced lower levels of IL-6 and VEGF, but higher levels of IGF-1 under basal conditions, as well as after stimulation with TNF, but not LPS. Neonatal and adult BMSCs had similar pluripotent potentials and cell surface markers, but nBMSCs proliferated faster. Furthermore, p38 and ERK appeared to play a more substantial role in nBMSC cytokine and growth factor production. Neonatal stem cells may aid in decreasing the local inflammatory response during ischemia, and could possibly be expanded more rapidly than adult cells prior to therapeutic use.