scholarly journals Short-Term Cryopreservation and Thawing have Minimal Effects on Plasmodium Falciparum Ex Vivo Invasion Profile

2021 ◽  
Author(s):  
Laty G. Thiam ◽  
Felix Ansah ◽  
Makhtar Niang ◽  
Gordon Awandare ◽  
Yaw Aniweh
Keyword(s):  
Ex Vivo ◽  
Clinical Isolates ◽  
Short Term ◽  
Culture Adaptation ◽  
Erythrocyte Invasion ◽  
Laboratory Facilities ◽  
Reliable Mechanism ◽  
Almost All ◽  
Selection Of

Abstract Ex vivo phenotyping of P. falciparum erythrocyte invasion diversity is important in the identification and down selection of potential malaria vaccine targets. However, due to the lack of appropriate laboratory facilities in remote areas of endemic countries, direct processing of P. falciparum clinical isolates is usually not feasible. Here, we investigated the combined effect of short-term cryopreservation and thawing processes on the ex vivo invasion phenotypes of P. falciparum isolates. Ex-vivo or in vitro invasion phenotyping assays were performed with P. falciparum clinical isolates prior to or following culture adaptation, respectively. All isolates were genotyped at Day 0 for parasite clonality. Subsequently, isolates that were successfully culture-adapted were genotyped again at Days 7, 15, 21, and 28-post adaptation. Invasion phenotyping assays were performed in isogenic isolates revived at different time points (3, 6, and 12 months) post-cryopreservation and the resulting data were compared to that from ex-vivo invasion data of matched isogenic parental isolates. Our findings indicate that natural P. falciparum infections mostly occur as polyclonal infections. We also show that short-term culture adaptation selects for parasite clonality and could be a driving force for variation in invasion phenotypes as compared to ex vivo data where almost all parasite clones of a given isolate are present. Interestingly, our data show little variation in the parasites’ invasion phenotype following short-term cryopreservation. Altogether, our data suggest that short-term cryopreservation of uncultured P. falciparum clinical isolates is a reliable mechanism for storing parasites for future use.

IN VITRO METHODS FOR THE STUDY OF THE ADENOHYPOPHYSIAL FUNCTIONS TO SECRETE GONADOTROPHIN

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S27-S40 ◽  
Author(s):  
T. Kobayashi ◽  
T. Kigawa ◽  
M. Mizuno ◽  
T. Watanabe
Keyword(s):  
Cell Culture ◽  
Organ Culture ◽  
Cultured Cells ◽  
Cell Sheet ◽  
Short Term ◽  
Hypothalamic Extract ◽  
Numerous Cell ◽  
Single Cell Culture ◽  
Almost All

ABSTRACT There are several in vitro methods to analyse the function of the adenohypophysis or the mechanisms of its regulation. The present paper deals with single cell culture, organ culture and short term incubation techniques by which the morphology and gonadotrophin-secreting function of the adenohypophysis were studied. In trypsin-dispersed cell culture, the adenohypophysial cells showed extensive propagation to form numerous cell colonies and finally develop into a confluent monolayer cell sheet covering completely the surface of culture vessels. Almost all of the cultured cells, however, became chromophobic, at least at the end of the first week of cultivation, when gonadotrophin was detectable neither in the culture medium nor in the cells themselves. After the addition of the hypothalamic extract, gonadotrophin became detectable again, and basophilic or PAS-positive granules also reappeared within the cells, suggesting that the gonadotrophs were stimulated by the extract to produce gonadotrophin. In organ culture and short term incubation, the incorporation of [3H] leucine into the adenohypophysial cells in relation to the addition of hypothalamic extract was examined. It was obvious that the ability to incorporate [3H] leucine into the gonadotrophs in vitro was highly dependent upon the presence of the hypothalamic extract.

The Effects of Shock Vancomycin Concentrations on the Formation of Heteroresistance in Staphylococcus aureus

2020 ◽  
Vol 65 (9-10) ◽  
pp. 3-7
Author(s):  
V. V. Gostev ◽  
Yu. V. Sopova ◽  
O. S. Kalinogorskaya ◽  
M. E. Velizhanina ◽  
I. V. Lazareva ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
In Vitro Selection ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
High Concentration ◽  
Short Term ◽  
High Concentrations ◽  
Selection Of ◽  
Selection Of Resistance ◽  
Test Cultures

Glycopeptides are the basis of the treatment of infections caused by MRSA (Methicillin-Resistant Staphylococcus aureus). Previously, it was demonstrated that antibiotic tolerant phenotypes are formed during selection of resistance under the influence of high concentrations of antibiotics. The present study uses a similar in vitro selection model with vancomycin. Clinical isolates of MRSA belonging to genetic lines ST8 and ST239, as well as the MSSA (ATCC29213) strain, were included in the experiment. Test isolates were incubated for five hours in a medium with a high concentration of vancomycin (50 μg/ml). Test cultures were grown on the medium without antibiotic for 18 hours after each exposure. A total of ten exposure cycles were performed. Vancomycin was characterized by bacteriostatic action; the proportion of surviving cells after exposure was 70–100%. After selection, there was a slight increase in the MIC to vancomycin (MIC 2 μg/ml), teicoplanin (MIC 1.5–3 μg/ml) and daptomycin (MIC 0.25–2 μg/ml). According to the results of PAP analysis, all strains showed an increase in the area under curve depending on the concentration of vancomycin after selection, while a heteroresistant phenotype (with PAP/AUC 0.9) was detected in three isolates. All isolates showed walK mutations (T188S, D235N, E261V, V380I, and G223D). Exposure to short-term shock concentrations of vancomycin promotes the formation of heteroresistance in both MRSA and MSSA. Formation of VISA phenotypes is possible during therapy with vancomycin.

scholarly journals Molecular Analysis of the gyrA and gyrB Quinolone Resistance-Determining Regions of Fluoroquinolone-Resistant Clostridium difficile Mutants Selected In Vitro

2009 ◽  
Vol 53 (6) ◽  
pp. 2463-2468 ◽  
Author(s):  
Patrizia Spigaglia ◽  
Fabrizio Barbanti ◽  
Thomas Louie ◽  
Frédéric Barbut ◽  
Paola Mastrantonio
Keyword(s):  
Clostridium Difficile ◽  
Clinical Isolates ◽  
Fluoroquinolone Resistance ◽  
Toxin B ◽  
Relevant Role ◽  
Vitro Development ◽  
Selection Of

ABSTRACT Recent studies have suggested that exposure to fluoroquinolones represents a risk factor for the development of Clostridium difficile infections and that the acquisition of resistance to the newer fluoroquinolones is the major reason facilitating wide dissemination. In particular, moxifloxacin (MX) and levofloxacin (LE) have been recently associated with outbreaks caused by the C. difficile toxinotype III/PCR ribotype 027/pulsed-field gel electrophoresis type NAP1 strain. In this study, we evaluated the potential of MX and LE in the in vitro development of fluoroquinolone resistance mediated by GyrA and GyrB alterations. Resistant mutants were obtained from five C. difficile parent strains, susceptible to MX, LE, and gatifloxacin (GA) and belonging to different toxinotypes, by selection in the presence of increasing concentrations of MX and LE. Stable mutants showing substitutions in GyrA and/or GyrB were obtained from the parent strains after selection by both antibiotics. Mutants had MICs ranging from 8 to 128 μg/ml for MX, from 8 to 256 μg/ml for LE, and from 1.5 to ≥32 μg/ml for GA. The frequency of mutation ranged from 3.8 × 10−6 to 6.6 × 10−5 for MX and from 1.0 × 10−6 to 2.4 × 10−5 for LE. In total, six different substitutions in GyrA and five in GyrB were observed in this study. The majority of these substitutions has already been described for clinical isolates or has occurred at positions known to be involved in fluoroquinolone resistance. In particular, the substitution Thr82 to Ile in GyrA, the most common found in resistant C. difficile clinical isolates, was observed after selection with LE, whereas the substitution Asp426 to Val in GyrB, recently described in toxin A-negative/toxin B-positive epidemic strains, was observed after selection with MX. Interestingly, a reduced susceptibility to fluoroquinolones was observed in colonies isolated after the first and second steps of selection by both MX and LE, with no substitution in GyrA or GyrB. The results suggest a relevant role of fluoroquinolones in the emergence and selection of fluoroquinolone-resistant C. difficile strains also in vivo.

scholarly journals TMC310911, a Novel Human Immunodeficiency Virus Type 1 Protease Inhibitor, ShowsIn Vitroan Improved Resistance Profile and Higher Genetic Barrier to Resistance Compared with Current Protease Inhibitors

2011 ◽  
Vol 55 (12) ◽  
pp. 5723-5731 ◽  
Author(s):  
Inge Dierynck ◽  
Herwig Van Marck ◽  
Marcia Van Ginderen ◽  
Tim H. M. Jonckers ◽  
Madhavi N. L. Nalam ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Protease Inhibitor ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Clinical Isolates ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Selection Of

ABSTRACTTMC310911 is a novel human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) structurally closely related to darunavir (DRV) but with improved virological characteristics. TMC310911 has potent activity against wild-type (WT) HIV-1 (median 50% effective concentration [EC50], 14 nM) and a wide spectrum of recombinant HIV-1 clinical isolates, including multiple-PI-resistant strains with decreased susceptibility to currently approved PIs (fold change [FC] in EC50, >10). For a panel of 2,011 recombinant clinical isolates with decreased susceptibility to at least one of the currently approved PIs, the FC in TMC310911 EC50was ≤4 for 82% of isolates and ≤10 for 96% of isolates. The FC in TMC310911 EC50was ≤4 and ≤10 for 72% and 94% of isolates with decreased susceptibility to DRV, respectively.In vitroresistance selection (IVRS) experiments with WT virus and TMC310911 selected for mutations R41G or R41E, but selection of resistant virus required a longer time than IVRS performed with WT virus and DRV. IVRS performed with r13025, a multiple-PI-resistant recombinant clinical isolate, and TMC310911 selected for mutations L10F, I47V, and L90M (FC in TMC310911 EC50= 16). IVRS performed with r13025 in the presence of DRV required less time and resulted in more PI resistance-associated mutations (V32I, I50V, G73S, L76V, and V82I; FC in DRV EC50= 258). The activity against a comprehensive panel of PI-resistant mutants and the limitedin vitroselection of resistant viruses under drug pressure suggest that TMC310911 represents a potential drug candidate for the management of HIV-1 infection for a broad range of patients, including those with multiple PI resistance.

scholarly journals FORMULATION AND OPTIMIZATION OF CELECOXIB NANOEMULGEL

2017 ◽  
Vol 10 (8) ◽  
pp. 353
Author(s):  
Sankha Bhattacharya ◽  
Bhupendra G Prajapati
Keyword(s):  
Ex Vivo ◽  
Skin Irritation ◽  
Scale Up ◽  
Pilot Scale ◽  
Water Soluble ◽  
Thaw Cycle ◽  
Diffusion Enhancement ◽  
Intrinsic Solubility ◽  
Almost All

Objective: The main objective of this experiment was to prepare and optimized celecoxib nanoemulgel. This formulation can be used for acuterheumatoid arthritis patients.Methods: Celecoxib is a poorly water soluble drug. We prepared celecoxib nanoemulgel to improve intrinsic solubility of celecoxib and enhancedeeper permeation throughout the skin. After several screening, the combination of acetonitrile, triacetin, campul 908P was considered for oil phase;acconon MC8-2EP as surfactant, and capmul MCM C-10 as a co-surfactant accordingly. As per Box-Behnken surface design model, optimization wasdone for all the 13 formulations.Results: Based on pseudo ternary plot, it was found that 4:1 Smix ratio was optimum and possessed maximum drug solubility. Further, screeningshown, 0.25-0.75% carbopol-940 can be a stable candidate for hydrogel preparation. Prepared nanoemulsions and hydrogels were admixed to preparenanoemulgel. Based on overlay plot, EG14* formulation was consider as optimum one, and various evaluation parameters were performed along withother formulations. Using Franz diffusion cell, in-vitro diffusion studies was performed. Almost all the formulations produces good qualitative drugrelease profile. The EG14* shown 95.50% drug release after 12th hrs with standard Higuchi plot (R2 value 0.9989). The optimum viscosity was foundto be 521±0.81 mPas at 100 rpm. The appearance of the formulations was milky, yellowish white with expectable pH ranged from 5.8 to 6.7. Theoptimized formulation has good spreadability coefficient, good ex-vivo diffusion enhancement factor (3.03) as compare to marketed gel. Mostly, ourformulations have less skin irritation and higher anti-inflammatory activity (92.56% of inhibition of paw edema for EG14*).Conclusion: From the thermodynamic studies, it was confirmed that EG14* maintained excellent stability profile in various heating-cooling cycle,centrifugation, and freeze-thaw cycle condition. Hence, it can be conclude that, our formulation, can be consider for pilot scale up.

scholarly journals Attenuation of Plasmodium falciparum in vitro drug resistance phenotype following culture adaptation compared to fresh clinical isolates in Cambodia

2015 ◽  
Vol 14 (1) ◽  
Author(s):  
Suwanna Chaorattanakawee ◽  
Charlotte A. Lanteri ◽  
Siratchana Sundrakes ◽  
Kritsanai Yingyuen ◽  
Panita Gosi ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasmodium Falciparum ◽  
Clinical Isolates ◽  
Resistance Phenotype ◽  
Culture Adaptation

scholarly journals A Comparison of Ex Vivo Expanded Human Regulatory T Cells Using Allogeneic Stimulated B Cells or Monocyte-Derived Dendritic Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Linda M. Lee ◽  
Hong Zhang ◽  
Karim Lee ◽  
Horace Liang ◽  
Alexander Merleev ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
B Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Low Frequencies ◽  
Almost All

Alloreactive regulatory T cells (arTregs) are more potent than polyclonal Tregs at suppressing immune responses to transplant antigens. Human arTregs can be expanded with allogeneic CD40L-stimulated B cells (sBcs) or stimulated-matured monocyte-derived dendritic cells (sDCs). Here, we compared the expansion efficiency and properties of arTregs stimulated ex vivo using these two types of antigen-presenting cells. Compared to sBcs, sDCs stimulated Tregs to expand two times more in number. The superior expansion-inducing capacity of sDCs correlated with their higher expression of CD80, CD86, and T cell-attracting chemokines. sBc- and sDC-arTregs expressed comparable levels of FOXP3, HELIOS, CD25, CD27, and CD62L, demethylated FOXP3 enhancer and in vitro suppressive function. sBc- and sDCs-arTregs had similar gene expression profiles that were distinct from primary Tregs. sBc- and sDC-arTregs exhibited similar low frequencies of IFN-γ, IL-4, and IL-17A-producing cells, and the cytokine-producing arTregs expressed high levels of FOXP3. Almost all sBc- and sDC-arTregs expressed CXCR3, which may enable them traffic to inflammatory sites. Thus, sDCs-arTregs that expand more readily, are phenotypically similar to sBc-arTregs, supporting sDCs as a viable alternative for arTreg production for clinical evaluation.

scholarly journals Amino Sugars Modify Antagonistic Interactions between Commensal Oral Streptococci andStreptococcus mutans

2019 ◽  
Author(s):  
Lulu Chen ◽  
Brinta Chakraborty ◽  
Jing Zou ◽  
Robert A. Burne ◽  
Lin Zeng
Keyword(s):  
Streptococcus Mutans ◽  
Ex Vivo ◽  
Arginine Deiminase ◽  
Clinical Isolates ◽  
Amino Sugars ◽  
Oral Streptococci ◽  
Streptococcus Gordonii ◽  
Oral Biofilms ◽  
Low Passage

ABSTRACTN-acetylglucosamine (GlcNAc) and glucosamine (GlcN) enhance the competitiveness of the laboratory strain DL1 ofStreptococcus gordoniiagainst the caries pathogenStreptococcus mutans. Here we examine how amino sugars affect the interaction of five low-passage clinical isolates of abundant commensal streptococci withS. mutansutilizing a dual-species biofilm model. Compared to glucose, growth on GlcN or GlcNAc significantly reduced the viability ofS. mutansin co-cultures with most commensals, shifting the proportions of species. Consistent with these results, production of H2O2was increased in most commensals when growing on amino sugars, and inhibition ofS. mutansbyStreptococcus cristatus, Streptococcus oralis,orS. gordoniiwas enhanced by amino sugars on agar plates. All commensals exceptS. oralishad higher arginine deiminase activities when grown on GlcN, and in some cases GlcNAc. Inex vivobiofilms formed using pooled cell-containing saliva (CCS), the proportions ofS. mutanswere drastically diminished when GlcNAc was the primary carbohydrate. Increased production of H2O2could account in large part for the inhibitory effects of CCS biofilms. Surprisingly, amino sugars appeared to improve mutacin production byS. mutanson agar plates, suggesting that the commensals have mechanisms to actively subvert antagonism byS. mutansin co-cultures. Collectively, these findings demonstrate that amino sugars can enhance the beneficial properties of low-passage commensal oral streptococci and highlight their potential for moderating the cariogenicity of oral biofilms.SIGNIFICANCEDental caries is driven by dysbiosis of oral biofilms in which dominance by acid-producing and acid-tolerant bacteria results in loss of tooth mineral. Our previous work demonstrated the beneficial effects of amino sugars, GlcNAc and GlcN, in promoting the antagonistic properties of a health-associated oral bacterium,Streptococcus gordonii,in competition with the major caries pathogenStreptococcus mutans.Here we investigated 5 low-passage clinical isolates of the most common streptococcal species to establish how amino sugars may influence the ecology and virulence of oral biofilms. Using multiplein vitromodels, including a human saliva-derived microcosm biofilm, experiments showed significant enhancement by at least one amino sugar in the ability of most of these bacteria to suppress the caries pathogen. Therefore, our findings demonstrated the mechanism of action by which amino sugars may affect human oral biofilms to promote health.

scholarly journals A Role for TNF-α in Chronic Lymphocytic Leukemia Bone Marrow Hematopoietic Dysfunction

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4276-4276
Author(s):  
Bryce A Manso ◽  
Jordan Krull ◽  
Kimberly Gwin ◽  
Petra Lothert ◽  
Charla R Secreto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Research Funding ◽  
Ex Vivo ◽  
Safety Monitoring ◽  
Data Safety Monitoring Board ◽  
Lymphocytic Leukemia ◽  
Cell Contact ◽  
Short Term ◽  
Tnf Α

The current paradigms of known peripheral immune abnormalities in B-Chronic Lymphocytic Leukemia (CLL) are not able to consistently explain patient complications and it is difficult to correct a given CLL patient's immune status. Here, we expand on our initial report demonstrating bone marrow (BM) hematopoietic dysfunction in untreated CLL patients (Manso et al., Leukemia volume 33, pages 638-652, 2019). CLL patient BM had significantly reduced frequencies and short-term functional capacity of hematopoietic stem and progenitor cells (HSPCs). Additionally, the remaining progenitors exhibited increased protein levels of the key hematopoietic transcriptional regulators GATA-2 and PU.1. We further evaluated the frequency and function of myeloid stem cells from controls and untreated CLL patients by limiting dilution analysis in long-term culture-initiating cell (LTC-IC) assays. Over the 5 week duration of the assay, we observed delayed and partial recovery of myelopoiesis from CLL-derived HSPCs (Figure 1A). These data suggest that removal of HSPCs from the CLL leukemic microenvironment partially recovers their ability to sustain myelopoiesis. A known inflammatory mediator and hematopoiesis-modulating cytokine that is constitutively produced by CLL cells, TNF-α, induced increased expression of GATA-2 and PU.1 in specific HSPC subsets and reduced formation of short-term colony forming units in vitro. Addition of TNF-α to LTC-IC assays resulted in a striking ablation of myelopoiesis in a dose-dependent manner, partially reproducing the ex vivo results (Figure 1B). To further assess the direct impact of CLL cells on HSPC biology, isolated HSPCs from controls were exposed in vitro to leukemic CLL cells. The co-culture induced overexpression of GATA-2 and PU.1 in distinct HSPC populations, recapitulating our ex vivo findings (Figure 1C-D). When cell-cell contact was inhibited by use of Transwell inserts, an intermediate increase in GATA-2 and PU.1 was observed, highlighting the contributions of both soluble mediators and cell-cell contact to HSPC alterations. In both direct and Transwell co-culture conditions, overexpression of GATA-2 and PU.1 was reversed when TNF-α was neutralized (Figure 1E-F). Taken together, these findings indicate a significant role for CLL-derived TNF-α in HSPC modulation and expand our previous observations of BM dysfunction in untreated CLL patients. This data offers new molecular insight into the contribution of the leukemic microenvironment to altered hematopoiesis, contributing to immunodeficiency in CLL, and identifies TNF-α as a potential therapeutic target for correction of hematopoiesis in CLL disease. Disclosures Ding: Merck: Research Funding; DTRM Biopharma: Research Funding. Parikh:AstraZeneca: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Pharmacyclics: Honoraria, Research Funding; Janssen: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria. Novak:Celgene Coorperation: Research Funding. Kay:Agios: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board.

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