Ultrastructural analysis of the invasion of tick cells by Lyme disease spirochetes (Borrelia burgdorferi) in vitro

1994 ◽  
Vol 72 (6) ◽  
pp. 977-994 ◽  
Author(s):  
T. J. Kurtti ◽  
U. G. Munderloh ◽  
S. F. Hayes ◽  
D. E. Krueger ◽  
G. G. Ahlstrand
Keyword(s):  
Electron Microscopy ◽  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Cell Lines ◽  
Cell Cultures ◽  
Dense Body ◽  
Tick Cell ◽  
Coated Pits ◽  
Intracellular Digestion

The association of Lyme disease spirochetes, Borrelia burgdorferi, with tick cell cultures was characterized by electron microscopy. These cells were active in endocytosis and intracellular digestion, containing coated vesicles, pits, and phagosomes. Borrelia burgdorferi in tick cell cultures resembled those described in tick tissues. In RAE25 cultures, isolated from Rhipicephalus appendiculatus embryos, invasion of cells was mediated by coated pits, indicating active host cell participation. The invading, tapered end of B. burgdorferi contained an electron-dense body that persisted throughout invasion. A host-derived membrane surrounded invading and intracellular borreliae as observed by transmission electron microscopy of cross and longitudinal sections, whereas degenerating ones lay inside secondary lysosomes. Borrelia burgdorferi cocultivated with cell lines from Ixodes scapularis were mainly found at the cell surface or within lysosomes. The differences seen in invasion of these cell lines are interpreted to reflect differences in cell types rather than species. Gemmae, indicative of degenerative changes in the spirochetes, were observed extra- and intra-cellularly. Membrane blebs were liberated by the spirochetes into the medium and onto the cells, and were avidly endocytosed. Tick cell cultures are a useful tool to elucidate spirochete – vector cell interactions that may be obscured in vivo.

scholarly journals Modulation of Borrelia burgdorferi Stringent Response and Gene Expression during Extracellular Growth with Tick Cells

2002 ◽  
Vol 70 (6) ◽  
pp. 3061-3067 ◽  
Author(s):  
Julia Bugrysheva ◽  
Elena Y. Dobrikova ◽  
Henry P. Godfrey ◽  
Marina L. Sartakova ◽  
Felipe C. Cabello
Keyword(s):  
Gene Expression ◽  
Borrelia Burgdorferi ◽  
Cell Lines ◽  
Mrna Level ◽  
Stringent Response ◽  
Global Regulator ◽  
Tick Cell ◽  
Fourfold Increase ◽  
Reciprocal Interactions

ABSTRACT Borrelia burgdorferi N40 multiplied extracellularly when it was cocultured with tick cells in L15BS medium, a medium which by itself did not support B. burgdorferi N40 growth. Growth of B. burgdorferi N40 in the presence of tick cells was associated with decreased production of (p)ppGpp, the stringent response global regulator, a fourfold decrease in relA/spoT mRNA, an eightfold net decrease in bmpD mRNA, and a fourfold increase in rpsL-bmpD mRNA compared to growth of B. burgdorferi in BSK-H medium. As a result, the polycistronic rpsL-bmpD mRNA level increased from 3 to 100% of the total bmpD message. These observations demonstrate that there are reciprocal interactions between B. burgdorferi and tick cells in vitro and indicate that the starvation-associated stringent response mediated by (p)ppGpp present in B. burgdorferi growing in BSK-H medium is ameliorated in B. burgdorferi growing in coculture with tick cell lines. These results suggest that this system can provide a useful model for identifying genes controlling interactions of B. burgdorferi with tick cells in vitro when it is coupled with genetic methods to isolate and complement B. burgdorferi mutants.

Attachment, entry, and destruction of cultured human B-lymphocytes by the lyme-disease spirochete borrelia burgdorferi

1996 ◽  
Vol 54 ◽  
pp. 808-809
Author(s):  
Elizabeth R. Fischer ◽  
David W. Dorward
Keyword(s):  
Electron Microscopy ◽  
Immune System ◽  
B Cells ◽  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
The United States ◽  
Host Cells ◽  
Interference Microscopy ◽  
Low Passage

The most prevalent tick-borne disease in the United States, Lyme disease, is caused by the spirochete Borrelia burgdorferi. Studies have suggested that Lyme disease spirochetes, like other bacteria, may enter host cells thereby evading the immune system.In order to investigate possible interactions between Lyme disease spirochetes and immune system cells, we co-incubated B. burgdorferi strain Sh-2-82 with cultured human B- and T-cells (TIB-215 and H9, ATCC, respectively) and primary lymphocytes at an MOI of 100:1.Nomarski interference microscopy revealed that as early as 10 minutes after coincubation, as previously described with other cell lines, low passage spirochetes (<8 in vitro passages) attached tip first to the B-cells and then invaded the B-cells (Fig. 1,4). Scanning electron microscopy revealed that by 24 hours post-infection, 90+% of B-cells had spirochetes attached to their surfaces (Fig.2). Transmission electron microscopy suggested that intracellular spirochetes may exist both in vacuoles and free in the cytoplasm of primary lymphocytes (Fig. 3a-b).

Hepatoma Cell Cultures as In Vitro Models for the Hepatotoxicity of Xenobiotics

1988 ◽  
Vol 16 (1) ◽  
pp. 32-37
Author(s):  
Margherita Ferro ◽  
Anna Maria Bassi ◽  
Giorgio Nanni
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Membrane Lipids ◽  
Hepatoma Cell ◽  
Positive Control ◽  
In Vitro Models ◽  
Low Concentrations ◽  
Formation Efficiency ◽  
Dose Dependent

Two hepatoma cell cultures were examined as in vitro models to be used in genotoxicity and cytotoxicity tests without the addition of bioactivating enzymes. The MH1C1, and HTC hepatoma lines were used in this study to establish their sensitivity to a number of xenobiotics, namely, cyclophosphamide (CP), the classical positive control in bioactivation tests; benzaldehyde (BA), a short-chain aldehyde; and 4-hydroxynonenal (HNE), a major toxic end-product of the peroxidative degradation of cell membrane lipids. As a first approach, we compared the following cytotoxicity tests: release of lactate dehydrogenase (LDH), and colony formation efficiency (CF). Colony-forming cells were exposed to the drugs according to different procedures, before or after the anchorage phase. The leakage of LDH into the medium following exposure of both cell lines to HNE, CP and BA for up to 24 hours was found not to be a good index of cytotoxicity. A better indicator of cytotoxicity was CF, as evaluated by exposure of the cells 24 hours after seeding. The effects were detectable at very low concentrations, corresponding to 10, 90 and 100μM for HNE, CP and BA, respectively. The impairment of CF efficiency was dose-dependent and time-dependent, and several differences between the two cell lines were observed.

scholarly journals Isolation and initial propagation of guinea pig adenovirus (GPAdV) in Cavia porcellus cell lines

F1000Research ◽  
2020 ◽  
Vol 8 ◽  
pp. 1597
Author(s):  
Adriana E. Kajon ◽  
Xiaoxin Li ◽  
Gabriel Gonzalez ◽  
Susan Core ◽  
Helga Hofmann-Sieber ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Cell Lines ◽  
Cell Cultures ◽  
Sequence Data ◽  
Respiratory Illness ◽  
Epithelial Cell Line ◽  
Cavia Porcellus ◽  
Diagnostic Assays ◽  
Tracheal Epithelial

Background:  The lack of adequate in vitro systems to isolate and propagate guinea pig adenovirus (GPAdV), a prevalent cause of respiratory illness of varaible severity in laboratory guinea pig colonies worldwide, has precluded its formal characterization to allow for the development of comprehensive diagnostic assays, and for the execution of complex pathogenesis and basic virology studies. Methods: Two strains of GPAdV were isolated in guinea pig (Cavia porcellus) cell cultures from frozen archival infected animal tissue originated from colony outbreaks of pneumonia in Australia and the Czech Republic in 1996. Results: Commercially available guinea pig cell lines from colorectal carcinoma (GPC-16), fetal fibroblast (104-C1) and lung fibroblast (JH4 C1), and the tracheal epithelial cell line GPTEC-T developed in this study were able to support viral infection and early propagation. Sufficient viral DNA was recovered from cell cultures to PCR-amplify and obtain sequence data for the complete hexon gene and partial DNA polymerase and penton base genes. Phylogenetic analysis for the three regions of the genome provided strong evidence confirming GPAdV as a unique species in the genus Mastadenovirus. Conclusions: This study demonstrated the feasibility of propagating GPAdV in cultures of immortalized lines of GP cells of a variety of types, thus establishing a critical foundation for the development of a robust culture platform for virus stock production and titration. The generation and analysis of whole GPAdV genome sequences will provide additional data for a comprehensive description of the genetic organization of the viral genome and for a better assessment of genetic diversity between the two isolated strains.

Variances in the Expression Profile of the EMT-Related Genes in Endometrial Cancer Lines In Vitro Study

2021 ◽  
Vol 22 ◽  
Author(s):  
Robert Nowakowski ◽  
Beniamin Grabarek ◽  
Anna Burnat-Olech ◽  
Dariusz Boroń ◽  
Monika Paul-Samojedny
Keyword(s):  
Endometrial Cancer ◽  
Expression Pattern ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cell Cultures ◽  
Expression Profile ◽  
Histological Grade ◽  
Cancer Cell Lines ◽  
Endometrial Cancer Cell

Background: This study aimed to evaluate the variances in the expression pattern of mRNAs and miRNAs related to the EMT in the Ishikawa (histological grade 1; G1), EC-1A (histological grade 2; G2), and KLE (histological grade 3; G3) cell cultures under cisplatin treatment. Methods: Endometrial cancer cell lines were exposed to 75.22 mg (an average concentration of the drug used in patients with endometrial cancer) for 12.24 and 48 hours compared to the untreated cells (control). The molecular analysis included extraction of total RNA, microarray analysis (mRNA and miRNA), RTqPCR, and the ELISA assay. Results: Out of 226 mRNAs associated with the EMT, the number of mRNAs differentially expressed in endometrial cancer cell cultures treated with cisplatin compared to a control culture was as follows: Ishikawa line - 87 mRNAs; EC-1A - 84 mRNAs; KLE - 71 mRNAs (p<0.05). The greatest changes in the Ishikawa line treated with the drug compared to the control were noticed for mRNA STAT1 TGFβ1, SMAD3, FOXO8, whereas in EC-1A, they were mRNA TGFβ1, BAMBI, SMAD4, and in KLE mRNA COL1A1, FOXO8, TGFβ1. The analysis also showed that miR-106a, miR-30d, miR-300 are common for all cell lines used in this experiment. Conclusion: Cisplatin changes the expression profile of genes associated with EMT in endometrial cancer cell lines. It seems that the expression pattern of TGFβ1 might be a promising, supplementary molecular marker of the effectiveness of cisplatin therapy. The analysis showed that miR-30d, miR-300, and miR-106a are involved in the regulation of the expression of EMT-related genes.

scholarly journals The R-SNARE Endobrevin/VAMP-8 Mediates Homotypic Fusion of Early Endosomes and Late Endosomes

2000 ◽  
Vol 11 (10) ◽  
pp. 3289-3298 ◽  
Author(s):  
Wolfram Antonin ◽  
Claudia Holroyd ◽  
Ritva Tikkanen ◽  
Stefan Höning ◽  
Reinhard Jahn
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Subcellular Fractionation ◽  
Immunogold Electron Microscopy ◽  
Fusion Reactions ◽  
Coated Pits ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Golgi Network

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of thetrans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

scholarly journals Cytotoxic Effect of Trabectedin In Human Adrenocortical Carcinoma Cell Lines and Primary Cells

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 928 ◽  
Author(s):  
Andrea Abate ◽  
Elisa Rossini ◽  
Sara Anna Bonini ◽  
Martina Fragni ◽  
Deborah Cosentini ◽  
...  
Keyword(s):  
Cytotoxic Activity ◽  
Cell Lines ◽  
Cell Cultures ◽  
Adrenocortical Carcinoma ◽  
Cytotoxic Effect ◽  
Alkylating Agents ◽  
Primary Cell ◽  
Primary Cell Cultures ◽  
High Cytotoxicity

Mitotane is the only drug approved for the treatment of adrenocortical carcinoma (ACC). The regimen to be added to mitotane is a chemotherapy including etoposide, doxorubicin, and cisplatin. This pharmacological approach, however, has a limited efficacy and significant toxicity. Evidence indicates that ACC seems to be sensitive to alkylating agents. Trabectedin is an anti-tumor drug that acts as an alkylating agent with a complex mechanism of action. Here, we investigated whether trabectedin could exert a cytotoxic activity in in vitro cell models of ACC. Cell viability was evaluated by MTT assay on ACC cell lines and primary cell cultures. The gene expression was evaluated by q-RT-PCR, while protein expression and localization were studied by Western blot and immunocytochemistry. Combination experiments were performed to evaluate their interaction on ACC cell line viability. Trabectedin demonstrated high cytotoxicity at sub-nanomolar concentrations in ACC cell lines and patient-derived primary cell cultures. The drug was able to reduce /β catenin nuclear localization, although it is unclear whether this effect is involved in the observed cytotoxicity. Trabectedin/mitotane combination exerted a synergic cytotoxic effect in NCI-H295R cells. Trabectedin has antineoplastic activity in ACC cells. The synergistic cytotoxic activity of trabectedin with mitotane provides the rationale for testing this combination in a clinical study.

scholarly journals Function of theBorrelia burgdorferiFtsH Homolog Is Essential for Viability bothIn VitroandIn Vivoand Independent of HflK/C

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Chen-Yi Chu ◽  
Philip E. Stewart ◽  
Aaron Bestor ◽  
Bryan Hansen ◽  
Tao Lin ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Membrane Proteins ◽  
Borrelia Burgdorferi ◽  
Cellular Response ◽  
Protein Transport ◽  
Protein Quality ◽  
Laboratory Culture ◽  
Biologically Active ◽  
Infectious Cycle

ABSTRACTIn many bacteria, the FtsH protease and its modulators, HflK and HflC, form a large protein complex that contributes to both membrane protein quality control and regulation of the cellular response to environmental stress. Both activities are crucial to the Lyme disease pathogenBorrelia burgdorferi, which depends on membrane functions, such as motility, protein transport, and cell signaling, to respond to rapid changes in its environment. Using an inducible system, we demonstrate that FtsH production is essential for both mouse and tick infectivity and forin vitrogrowth ofB. burgdorferi. FtsH depletion inB. burgdorfericells resulted in membrane deformation and cell death. Overproduction of the protease did not have any detectable adverse effects onB. burgdorferigrowthin vitro, suggesting that excess FtsH does not proteolytically overwhelm its substrates. In contrast, we did not observe any phenotype for cells lacking the protease modulators HflK and HflC (ΔHflK/C), although we examined morphology, growth rate, growth under stress conditions, and the complete mouse-tick infectious cycle. Our results demonstrate that FtsH provides an essential function in the life cycle of the obligate pathogenB. burgdorferibut that HflK and HflC do not detectably affect FtsH function.IMPORTANCELyme disease is caused byBorrelia burgdorferi, which is maintained in nature in an infectious cycle alternating between small mammals andIxodesticks.B. burgdorferiproduces specific membrane proteins to successfully infect and persist in these diverse organisms. We hypothesized thatB. burgdorferihas a specific mechanism to ensure that membrane proteins are properly folded and biologically active when needed and removed if improperly folded or dysfunctional. Our experiments demonstrate that FtsH, a protease that fulfills this role in other microorganisms, is essential toB. burgdorferiviability. Cells depleted of FtsH do not survive in laboratory culture medium and cannot colonize mice or ticks, revealing an absolute requirement for this protease. However, the loss of two potential modulators of FtsH activity, HflK and HflC, does not detectably affectB. burgdorferiphysiology. Our results provide the groundwork for the identification of FtsH substrates that are critical for the bacterium’s viability.

scholarly journals Initiation of primary cell cultures from embryonic Haemaphysalis bispinosa ticks 

2017 ◽  
Vol 22 (3) ◽  
pp. 323 ◽  
Author(s):  
Fang-shiang Lim ◽  
Jing-jing Khoo ◽  
Fezshin Chen ◽  
Lesley Bell-sakyi ◽  
Chee-sieng Khor ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Embryonic Cell ◽  
Rrna Gene ◽  
Hard Tick ◽  
Tick Cell ◽  
Haemaphysalis Bispinosa ◽  
Ornithodoros Moubata ◽  
Floating Cell ◽  
Axenic Conditions

Tick cell cultures have been widely used as an important tool for the study of tick-associated microorganisms, specifically for medically important bacteria or viruses that may be difficult to isolate or culture in axenic conditions. In this study, primary embryonic tick cell cultures were initiated separately from each of the egg batches laid by 10 female ticks belonging to the hard tick genus Haemaphysalis. All cultures were maintained at 28°C. After 10 months, 4 healthy cultures were identified with the potential for developing into continuous tick cell lines. These cultures comprise large cells predominantly forming floating cell clumps with multicellular vesicles, which are morphologically similar to cell lines derived from the soft tick Ornithodoros moubata. Subculture has not yet been performed due to the low cell density at the time of writing. Amplification and sequencing of a fragment of the 16S rRNA gene from DNA extracted from the parent ticks showed 99%-100% similarity to published sequences of Haemaphysalis bispinosa. This is the first report of the initiation of embryonic cell cultures from Haemaphysalis ticks found in Malaysia. Such tick cell cultures will be useful for studies of tick-borne pathogens in this region, where recent studies have shown that Haemaphysalis ticks are highly represented and harbor medically important bacteria. 

scholarly journals Molluscan cells in culture: primary cell cultures and cell lines

2013 ◽  
Vol 91 (6) ◽  
pp. 391-404 ◽  
Author(s):  
T.P. Yoshino ◽  
U. Bickham ◽  
C.J. Bayne
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cell Cultures ◽  
Snail Host ◽  
Primary Cell ◽  
Neoplastic Disease ◽  
Freshwater Bivalve ◽  
Primary Cell Cultures ◽  
Organ Systems

In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata (Say, 1818) embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host – parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome.

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