In vitro and in vivo effects of rifabutin alone or combined with atovaquone against Toxoplasma gondii.

The efficacy of rifabutin (RIFA) alone or in combination with atovaquone (ATO) was examined in vitro and in a murine model of acute toxoplasmosis. In vitro studies were performed with MRC5 fibroblast tissue cultures, with quantification of Toxoplasma growth by enzyme-linked immunosorbent assay. For in vivo studies, mice were acutely infected with 10(4) tachyzoites of the virulent RH strain and were then treated perorally for 10 days from day 1 or day 4 postinfection. The efficacy of each drug regimen was assessed by determination of survival rates and sequential titration of parasites in blood, brain, and lungs by a tissue culture method. In vitro, RIFA was inhibitory for Toxoplasma growth at concentrations between 0.5 and 20 micrograms/ml; the 50% inhibitory concentration was estimated to be 1.68 micrograms/ml. When RIFA and ATO were combined, synergistic effects were noted for RIFA at 20 micrograms/ml combined with ATO at 0.01 or 0.02 microgram/ml and RIFA at 1, 2, or 5 micrograms/ml combined with ATO at 0.02 microgram/ml. In vivo, administration of RIFA at 200 mg/kg of body weight per day from day 1 to day 10 resulted in a 100% protection during treatment, with clearance of parasites from the blood, brain, and lungs. After the cessation of therapy, relapses occurred in the brain and lungs; the mortality was 46% at the end of the experiment (day 30). Among the mice treated with RIFA at 200 mg/kg/day from day 4 to day 14, no death was recorded during the treatment period and a marked reduction in parasite burdens was observed in blood and tissues; however, relapses occurred and 10% of mice survived until day 30. Administration of RIFA at 200 mg/kg/day in combination with ATO at 100 mg/kg/day resulted in a marked prolongation of survival compared with that for mice that received ATO or RIFA alone. However, in mice receiving the combination, parasite burdens in blood and organs were similar to those in mice treated with RIFA alone. These results confirmed the activity of RIFA in the treatment of acute toxoplasmosis and the potential of the combination of RIFA-ATO since the two drugs act synergistically against Toxoplasma gondii.

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Recombinant Bactericidal/Permeability-Increasing Protein (rBPI21) in Combination with Sulfadiazine Is Active against Toxoplasma gondii

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.4.758 ◽

1999 ◽

Vol 43 (4) ◽

pp. 758-762 ◽

Cited By ~ 10

Author(s):

Anis A. Khan ◽

Lewis H. Lambert ◽

Jack S. Remington ◽

Fausto G. Araujo

Keyword(s):

Toxoplasma Gondii ◽

Synergistic Effects ◽

Day Treatment ◽

Protozoan Parasite ◽

Time To Death ◽

Significant Survival ◽

Mathematical Analyses ◽

Acute Toxoplasmosis

ABSTRACT The activity of recombinant bactericidal/permeability-increasing protein (rBPI21), alone or in combination with sulfadiazine, on the intracellular replication of Toxoplasma gondii was assessed in vitro and in mice with acute toxoplasmosis. rBPI21 markedly inhibited the intracellular growth of T. gondii in human foreskin fibroblasts (HFFs). Following 72 h of exposure, the 50% inhibitory concentration of rBPI21 for T. gondii was 2.6 μg/ml, whereas only slight cytotoxicity for HFF cells was observed at the concentrations tested. Subsequent mathematical analyses revealed that the combination of rBPI21 with sulfadiazine yielded slight to moderate synergistic effects against T. gondii in vitro. Infection of mice orally with C56 cysts or intraperitoneally (i.p.) with RH tachyzoites resulted in 100% mortality, whereas prolongation of the time to death or significant survival (P = 0.002) was noted for those animals treated with 5 to 20 mg of rBPI21 per kg of body weight per day. Treatment with rBPI21 in combination with sulfadiazine resulted in significant (P = 0.0001) survival of mice infected i.p. with tachyzoites but not of mice infected orally with T. gondii cysts. These results indicate that rBPI21 is active in vitro and in vivo against T. gondii and that its activity is significantly enhanced when it is used in combination with sulfadiazine. To our knowledge, this is the first report of the activity of rBPI21 against a protozoan parasite.

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Nutraceutical Activity in Osteoarthritis Biology: A Focus on the Nutrigenomic Role

Cells ◽

10.3390/cells9051232 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1232

Author(s):

Stefania D’Adamo ◽

Silvia Cetrullo ◽

Veronica Panichi ◽

Erminia Mariani ◽

Flavio Flamigni ◽

...

Keyword(s):

Signaling Pathways ◽

Antioxidant Activities ◽

Synergistic Effects ◽

National Health System ◽

Bioactive Molecules ◽

Joint Disease ◽

In Vivo Studies ◽

Wide Range

Osteoarthritis (OA) is a disease associated to age or conditions that precipitate aging of articular cartilage, a post-mitotic tissue that remains functional until the failure of major homeostatic mechanisms. OA severely impacts the national health system costs and patients’ quality of life because of pain and disability. It is a whole-joint disease sustained by inflammatory and oxidative signaling pathways and marked epigenetic changes responsible for catabolism of the cartilage extracellular matrix. OA usually progresses until its severity requires joint arthroplasty. To delay this progression and to improve symptoms, a wide range of naturally derived compounds have been proposed and are summarized in this review. Preclinical in vitro and in vivo studies have provided proof of principle that many of these nutraceuticals are able to exert pleiotropic and synergistic effects and effectively counteract OA pathogenesis by exerting both anti-inflammatory and antioxidant activities and by tuning major OA-related signaling pathways. The latter are the basis for the nutrigenomic role played by some of these compounds, given the marked changes in the transcriptome, miRNome, and methylome. Ongoing and future clinical trials will hopefully confirm the disease-modifying ability of these bioactive molecules in OA patients.

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Comparison of the synergistic effects of tamsulosin versus phentolamine on penile erection: in vitro and in vivo studies

Urological Research ◽

10.1007/s002400050133 ◽

1999 ◽

Vol 27 (6) ◽

pp. 437-444 ◽

Cited By ~ 1

Author(s):

S. C. Kim ◽

K. K. Seo ◽

S. K. Lee ◽

E. S. Song ◽

M. Y. Lee

Keyword(s):

Penile Erection ◽

Synergistic Effects ◽

In Vivo Studies

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Development of a System To Study CD4+-T-Cell Responses to Transgenic Ovalbumin-Expressing Toxoplasma gondii during Toxoplasmosis

Infection and Immunity ◽

10.1128/iai.72.12.7240-7246.2004 ◽

2004 ◽

Vol 72 (12) ◽

pp. 7240-7246 ◽

Cited By ~ 45

Author(s):

Marion Pepper ◽

Florence Dzierszinski ◽

Amy Crawford ◽

Christopher A. Hunter ◽

David Roos

Keyword(s):

T Cells ◽

T Cell ◽

Toxoplasma Gondii ◽

T Cell Responses ◽

Cell Receptor ◽

In Vivo Studies ◽

Cell Responses ◽

Ifn Γ

ABSTRACT The study of the immune response to Toxoplasma gondii has provided numerous insights into the role of T cells in resistance to intracellular infections. However, the complexity of this eukaryote pathogen has made it difficult to characterize immunodominant epitopes that would allow the identification of T cells with a known specificity for parasite antigens. As a consequence, analysis of T-cell responses to T. gondii has been based on characterization of the percentage of T cells that express an activated phenotype during infection and on the ability of these cells to produce cytokines in response to complex mixtures of parasite antigens. In order to study specific CD4+ T cells responses to T. gondii, recombinant parasites that express a truncated ovalbumin (OVA) protein, in either a cytosolic or a secreted form, were engineered. In vitro and in vivo studies reveal that transgenic parasites expressing secreted OVA are able to stimulate T-cell receptor-transgenic OVA-specific CD4+ T cells to proliferate, express an activated phenotype, and produce gamma interferon (IFN-γ). Furthermore, the adoptive transfer of OVA-specific T cells into IFN-γ−/− mice provided enhanced protection against infection with the OVA-transgenic (but not parental) parasites. Together, these studies establish the utility of this transgenic system to study CD4+-T-cell responses during toxoplasmosis.

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Low Intensity Pulsed Ultrasound for Bone Tissue Engineering

Micromachines ◽

10.3390/mi12121488 ◽

2021 ◽

Vol 12 (12) ◽

pp. 1488

Author(s):

Colleen McCarthy ◽

Gulden Camci-Unal

Keyword(s):

Bone Formation ◽

Bone Tissue ◽

Mechanical Stimulation ◽

Synergistic Effects ◽

In Vivo Studies ◽

Pulsed Ultrasound ◽

Low Intensity Pulsed Ultrasound ◽

Low Intensity

As explained by Wolff’s law and the mechanostat hypothesis, mechanical stimulation can be used to promote bone formation. Low intensity pulsed ultrasound (LIPUS) is a source of mechanical stimulation that can activate the integrin/phosphatidylinositol 3-OH kinase/Akt pathway and upregulate osteogenic proteins through the production of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). This paper analyzes the results of in vitro and in vivo studies that have evaluated the effects of LIPUS on cell behavior within three-dimensional (3D) titanium, ceramic, and hydrogel scaffolds. We focus specifically on cell morphology and attachment, cell proliferation and viability, osteogenic differentiation, mineralization, bone volume, and osseointegration. As shown by upregulated levels of alkaline phosphatase and osteocalcin, increased mineral deposition, improved cell ingrowth, greater scaffold pore occupancy by bone tissue, and superior vascularization, LIPUS generally has a positive effect and promotes bone formation within engineered scaffolds. Additionally, LIPUS can have synergistic effects by producing the piezoelectric effect and enhancing the benefits of 3D hydrogel encapsulation, growth factor delivery, and scaffold modification. Additional research should be conducted to optimize the ultrasound parameters and evaluate the effects of LIPUS with other types of scaffold materials and cell types.

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Assessing CD97 and CD99 As Markers of Leukaemia Initiating Cells in Paediatric ALL

Blood ◽

10.1182/blood.v120.21.1882.1882 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1882-1882 ◽

Cited By ~ 1

Author(s):

Charlotte Victoria Cox ◽

Paraskevi Diamanti ◽

Allison Blair

Keyword(s):

Functional Capacity ◽

Conflicts Of Interest ◽

Residual Disease ◽

Survival Rates ◽

In Vivo Studies ◽

Nsg Mice ◽

Early Treatment Response ◽

Current Therapies

Abstract Abstract 1882 Overall survival rates in paediatric acute lymphoblastic leukaemia (ALL) have dramatically improved but around 20% do not respond to current therapies and subsequently relapse. Leukaemia initiating cells (LIC) are the topic of much investigation, as these cells can self-renew and may have the potential to cause relapse. It has been shown that multiple subpopulations of ALL cells have the ability to initiate the disease in immune deficient mouse models. Therefore, treatment should be targeted at all cells with this capacity, if the disease is to be eradicated. Minimal residual disease (MRD) detection is an invaluable tracking tool to assess early treatment response and recent studies have highlighted potential markers that may improve the sensitivity of MRD detection by flow cytometry. CD97 and CD99 are two markers which were over expressed in paediatric ALL. Incorporating these markers into investigations of LIC may allow discrimination of leukaemia cells from normal haemopoietic stem cells (HSC). In this study we evaluated the expression of CD34 in combination with CD97 in B cell precursor (BCP) ALL cases and CD99 in T-ALL cases and subsequently assessed the functional capacity of the sorted subpopulations in vitro and in vivo. Ten ALL samples (6 B-ALL & 4 T-ALL) with a median age 7 years (range 2–15 years) were studied. One B-ALL case and 3 T-ALL cases were considered high risk by molecular assessment of MRD at day 28 of treatment. Flow cytometric analyses of the ALL samples and 8 normal haemopoietic cell samples demonstrated that both CD97 and CD99 were over expressed in ALL patients (78.9±14.8% & 76.4±32.8%, respectively) when compared to normal haemopoietic cells (14.1±25.4%; p=0.001, 47.1±10%; p=0.03, respectively). Cells were sorted for expression/lack of expression of these markers and proliferation of the sorted cells was assessed in suspension culture over a 6 week period. In the B-ALL patients the CD34+/CD97+ subpopulation represented the bulk of leukaemia cells (65.2±32.1%), the CD34−/CD97+ the smallest fraction (3.3±2.4%) with the CD34+/CD97− and CD34−/CD97− subpopulations representing 21.1±31.5% and 10.5±5.8% of cells, respectively. When the functional capacity of these subpopulations was assessed in vitro greatest expansion was observed in cells derived from CD34+/CD97− subpopulation (2–173 fold) from 9.4×103 at initiation up to 1.5×106 cells at week 6. Expansion was also observed, to a lesser extent in the CD34−/CD97− subpopulation (3.4–28 fold) from 8×103 up to 1.4×106 cells. No expansion was observed in cultures of CD34+/CD97+ and CD34−/CD97− subpopulations but cells were maintained throughout the culture period. These sorted subpopulations were also inoculated into NOD/LtSz-SCID IL-2Rγc null (NSG) mice to evaluate repopulating capacity. To date, engraftment has been achieved with 3 subpopulations; CD34+/CD97+ (3–28.8% CD45+), CD34+/CD97− (0.5–25.5% CD45+) and CD34−/CD97+ (23.8% CD45+) cells. When the functional capacity of T-ALL cases was assessed the CD34+/CD99+ subpopulation represented the bulk of cells at sorting (51.87±47.2%), the CD34+/CD99- subpopulation was the smallest (0.9±0.8%) and the CD34−/CD99+ and CD34−/CD99− subpopulations represented 32.1±38.9% and 27.2±33.4% of cells, respectively. Greatest expansion was observed in cultures of CD34+/CD99- cells (4.6–1798 fold) from 7.5×103 up to 2.6×106 cells at week 6. The other 3 subpopulations expanded to a lesser extent (1.3–216 fold) from 5×103 up to 1.8×106 cells. When the functional capacity of these cells was assessed in NSG mice, engraftment was achieved in all subpopulations; CD34+/CD99+ (87–90.5% CD45+), CD34+/CD99− (1.5–84.9% CD45+), CD34−/CD99+ (31.3–98.6% CD45+) and CD34−/CD99− (3–92.9% CD45+). In some cases, cells recovered from BM of NSG inoculated with CD99− cells had high expression of CD99, typical of the patient samples at diagnosis, indicating that the inoculated CD99− cells had differentiated in vivo. Studies are ongoing to assess the self-renewal capacity of these subpopulations by serial transplantation. The findings to date indicate that targeting CD97 and CD99, either alone or in combination with CD34 would not eliminate all cells with the capacity to initiate and maintain B-ALL and T-ALL, respectively. Further developments in therapy may require targeting leukaemogenic pathways, rather than only cell surface markers to improve survival outcome in paediatric ALL. Disclosures: No relevant conflicts of interest to declare.

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Histone deacetylase SIR2 in Toxoplasma gondii modulates functions of murine macrophages in vitro and protects mice against acute toxoplasmosis in vivo

Microbial Pathogenesis ◽

10.1016/j.micpath.2021.104835 ◽

2021 ◽

Vol 154 ◽

pp. 104835

Author(s):

ZhengQing Yu ◽

SiYing Chen ◽

MuhammadTahir Aleem ◽

SuHui He ◽

Yang Yang ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Histone Deacetylase ◽

Murine Macrophages ◽

Acute Toxoplasmosis

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EXTH-66. NEO100 TRANSIENTLY OPENS UP THE BLOOD BRAIN BARRIER VIA TIGHT JUNCTION INHIBITION

Neuro-Oncology ◽

10.1093/neuonc/noaa215.420 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii101-ii102

Author(s):

Thomas Chen ◽

Weijun Wang ◽

Nagore Marin Ramos ◽

Axel Schonthal

Keyword(s):

Tight Junction ◽

Blood Brain Barrier ◽

In Vitro Models ◽

Brain Barrier ◽

In Vivo Studies ◽

Blue Dye ◽

Blood Brain ◽

Main Technique

Abstract The blood brain barrier (BBB) prevents effective entry of nearly all therapeutics to the central nervous system (CNS), preventing effective treatment of brain-related malignancies. Intracarotid mannitol injection has been the main technique to transiently open up the BBB, with its attendant variability and complications. A more direct and better tolerated method is needed to open up the BBB. We present our discovery that intraarterial (IA) injection of NEO100, a cGMP-quality form of perillyl alcohol (POH), transiently opens up the BBB in a safe and reversible manner. We used in-vitro models of MDCK1 and patient derived brain endothelial cell (BEC) + astrocyte barriers to determine that NEO100 increased FITC-antibody diffusion across the in-vitro BBB model and decreased trans-epithelial/endothelial electrical resistance (TEER). NEO100 effects on transcellular and paracellular pathways were studied using western blot, flow cytometry, HPLC, fluorescent probes, microarray analysis, and transmission electron microscopy. In-vivo studies were performed using ultrasound-guided intracardiac administration of NEO100 in mice with subsequent intravenous delivery of non-BBB permeable therapeutic agents. We determined that NEO100 transiently disrupts the transcellular pathway by permeabilizing BEC membranes, and the paracellular pathway via delocalization of tight junction proteins. In vivo IA NEO100 administration caused an effective dose- and time-dependent BBB permeabilization, which was reversible and well tolerated by the mice. This was evidenced by the spreading of Evans blue dye, and of therapeutics with different molecular weights, ie methotrexate, anti-PD-1 antibody, and CAR-T cells in the brain. Our results demonstrate that IA NEO100 is able to open the BBB in a controlled and reversible manner, allowing it to facilitate drug delivery to the CNS.

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In vitro 3D Spheroid Culture Developed on the Parafilm Surface Using HEK-293 Cells

Academic Perspective Procedia ◽

10.33793/acperpro.03.01.48 ◽

2020 ◽

Vol 3 (1) ◽

pp. 220-227

Author(s):

Erdal Eroğlu

Keyword(s):

Three Dimensional ◽

Culture Method ◽

In Vivo Studies ◽

Preclinical Research ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Hanging Drop Culture

Preclinical research to predict the effects of drugs and chemicals on humans is commonly carried out either by cell culture studies in vitro condition or on animals in vivo condition. While drug studies tested on cells cultured as a monolayer in plastic flasks are incompatible with realistic results, falsifying findings can also be achieved from in vivo studies performed on different species. In recent years, research on drug tests using spheroid cultures formed by growing cells in three-dimensional (3D) in vitro has attracted great interest. 3D spheroid structures are formed by growing the cells in a drop suspended on superhydrophobic surfaces. In this study, HEK-293 cells were investigated on parafilm surfaces displaying superhydrophobic properties by growing in 2 &micro;l volume using hanging drop culture method in terms of spheroid formation. Light microscopy images from spheroid structures were taken on different incubation days and the area of spheroids was measured using the ImageJ program. Our study holds important findings for a chip platform that can be developed for use in vitro drug tests.

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