Microfluidic approach to create three-dimensional tissue models for biofilm-related infection of orthopaedic implants.

With conventional in vitro culture methods, it is difficult to study complex interactions of host cells with pathogens and drugs in physiologically relevant microenvironments. To simulate orthopaedic implant-associated infection, a multi-channel microfluidic device was used to (1) observe in real-time the development of osteoblasts into three-dimensional (3D) tissue-like structures and (2) study how this development was influenced by phenotypes of Staphylococcus epidermidis. In the absence of bacteria, osteoblasts formed a confluent layer on the bottom channel surface, gradually migrated to the side and top surfaces, and formed calcified 3D nodular structures in 8 days. The delivery timing and concentration of an antibiotic were controlled to produce small colony variants, sessile biofilms, or dead cells of S. epidermidis. In the presence of the small colony variants, osteoblasts initially adhered, and spread, but were killed within 2 days. In contrast, the sessile biofilms and dead bacteria cells did not significantly interfere with the formation of tissue-like structures. The results suggest the possibility of creating in vitro tissue-biofilm-biomaterial interfaces and therefore 3D tissue models, as an entirely new method of studying biofilm-related infection of orthopaedic implants with physiological relevance.

Dissecting the role of the Tir:Nck and Tir:IRTKS/IRSp53 signalling pathways in vivo.

Attaching and effacing (A/E) lesions and actin polymerization, the hallmark of enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) infections, are dependent on the effector Tir. Phosphorylation of Tir(EPEC/CR) Y474/1 leads to recruitment of Nck and neural Wiskott-Aldrich syndrome protein (N-WASP) and strong actin polymerization in cultured cells. Tir(EPEC/CR) also contains an Asn-Pro-Tyr (NPY(454/1)) motif, which triggers weak actin polymerization. In EHEC the NPY(458) actin polymerization pathway is amplified by TccP/EspF(U), which is recruited to Tir via IRSp53 and/or insulin receptor tyrosine kinase substrate (IRTKS). Here we used C. rodentium to investigate the different Tir signalling pathways in vivo. Following infection with wild-type C. rodentium IRTKS, but not IRSp53, was recruited to the bacterial attachment sites. Similar results were seen after infection of human ileal explants with EHEC. Mutating Y471 or Y451 in Tir(CR) abolished recruitment of Nck and IRTKS respectively, but did not affect recruitment of N-WASP or A/E lesion formation. This suggests that despite their crucial role in actin polymerization in cultured cells the Tir:Nck and Tir:IRTKS pathways are not essential for N-WASP recruitment or A/E lesion formation in vivo. Importantly, wild-type C. rodentium out-competed the tir tyrosine mutants during mixed infections. These results uncouple the Tir:Nck and Tir:IRTKS pathways from A/E lesion formation in vivo but assign them an important in vivo role.

Cholesterol binding by the bacterial type III translocon is essential for virulence effector delivery into mammalian cells.

A ubiquitous early step in infection of man and animals by enteric bacterial pathogens like Salmonella, Shigella and enteropathogenic Escherichia coli (EPEC) is the translocation of virulence effector proteins into mammalian cells via specialized type III secretion systems (TTSSs). Translocated effectors subvert the host cytoskeleton and stimulate signalling to promote bacterial internalization or survival. Target cell plasma membrane cholesterol is central to pathogen-host cross-talk, but the precise nature of its critical contribution remains unknown. Using in vitro cholesterol-binding assays, we demonstrate that Salmonella (SipB) and Shigella (IpaB) TTSS translocon components bind cholesterol with high affinity. Direct visualization of cell-associated fluorescently labelled SipB and parallel immunogold transmission electron microscopy revealed that cholesterol levels limit both the amount and distribution of plasma membrane-integrated translocon. Correspondingly, cholesterol depletion blocked effector translocation into cultured mammalian cells by not only the related Salmonella and Shigella TTSSs, but also the more divergent EPEC system. The data reveal that cholesterol-dependent association of the bacterial TTSS translocon with the target cell plasma membrane is essential for translocon activation and effector delivery into mammalian cells.

Inhibition of Salmonella intracellular proliferation by non-phagocytic eucaryotic cells.

Salmonella typhimurium is an intracellular pathogen capable of proliferating within vacuolar compartments of non-phagocytic eucaryotic cells. This process has been shown to be essential for virulence in the mouse typhoid model (Leung and Finlay, Proc. Natl. Acad. Sci. USA, 88, 11470-11474, 1990). Here we present evidence that certain non-phagocytic eucaryotic cell lines, such as 3T3 (mouse fibroblasts) and NRK (rat fibroblasts) cells, are not permissive for S. typhimurium intracellular proliferation. Moreover, viability of intracellular bacteria residing within both cell types notably decreases at late postinfection times (72 h). These results clearly demonstrate that non-phagocytic eucaryotic cells are capable of destroying intracellular S. typhimurium. Experimentation with 3T3 and NRK cell lines might provide an appropriate in vitro model for identifying new bacterial and/or eucaryotic factors regulating Salmonella intracellular proliferation within vacuoles of the host eucaryotic cell.

Antisense oligodeoxynucleotides to c-jun inhibits proliferation of transformed NIH 3T3 cells induced by E5a of HPV-11.

E5a of HPV-11 is a transforming oncogene. Previously, we had shown that the E5a gene is required only for the initiation of transformation; c-jun might be involved in the maintenance of transformation. In this study, we exposed E5a transformed NIH 3T3 cells to antisense oligodeoxynucleotides complementary to the 24 nucleotides corresponding to the translation initiation site of the c-jun gene, and examined the effects of this treatment on cell proliferation. Results show that antisense c-jun oligodeoxynucleotides could repress c-jun production and inhibit cell proliferation in E5a transformed cells.

Rapid quantitation of recombinant retroviruses.

A method for measuring the concentration of recombinant retrovirus encoding the Escherichia coli LacZ gene is described. The assay is based on the quantitative measurement of beta-galactosidase activity in extracts of cells infected with the LacZ-encoding retrovirus. LacZ-encoded beta-galactosidase activity in transduced cells is measured using the colorimetric substrate, o-nitrophenyl beta-D-galactopyranoside (ONPG), and the results are read using an ELISA plate-reader. Because the entire assay is performed in a 96-well plate, large numbers of samples are easily measured, and the assay has the advantages of rapidity, precision, and ease of data collection. The assay was used to determine the optimal concentration of polybrene, a polycation known to enhance the infectivity of retroviruses, and can be used to evaluate other factors that affect infection, as well as the optimal conditions for production of the recombinant retrovirus.

Hoechst 33258 staining for detecting mycoplasma contamination in cell cultures: a method for reducing fluorescence photobleaching.

DNA fluorochrome staining with Hoechst 33258 bisbenzimide is commonly used for detection of mycoplasma contamination in cell cultures. Photobleaching of Hoechst 33258 is pronounced under the conditions of intense illumination, high magnification and resolution required for detection of mycoplasmas. To reduce photobleaching we investigated the effects of some antioxidant molecules, p-phenylenediamine (PPD), n-propyl gallate (NPG) and 1,4-diazabicyclo(2,2,2)octane (DABCO), which are known to reduce the fading rate of fluorescein. Mycoplasma-contaminated cell monolayers were stained with Hoechst 33258 and mounted in glycerol containing different amounts of antioxidant additives. The cells were examined in an epifluorescence microscope, and the emitted light intensity was recorded. Results showed that PPD and, to a lower degree, NPG, retarded the photobleaching of Hoechst 33258-stained cells, whereas DABCO was not effective. However, fluorescence half-life was increased about three-fold by NPG and almost 20-fold by PPD. The rate of fluorescence fading of Hoechst 33258 can therefore be retarded by PPD, with obvious advantages for reading and photographic recording of results.

The effect of GM-CSF and G-CSF on the growth of human osteosarcoma cells in vitro and in vivo.

The human osteosarcoma cell line, MG63, responds both to GM-CSF and to G-CSF in vitro. To assess the significance of these observations to tumor growth in vivo, MG63 cells were engineered by retroviral infection to produce human GM-CSF or G-CSF. These retrovirally infected cells become autostimulatory as measured by increased [3H]-thymidine incorporation (3- to 7-fold) and anchorage-independent colony formation (7- to 10-fold) as compared with uninfected MG63 cells or cells infected with control (neor) retrovirus. The increased proliferation induced by exogenous GM-CSF or G-CSF on uninfected MG63 cells in both assays could be completely inhibited by anti-GM-CSF or anti-G-CSF antibodies, while the same antibodies only partially abrogated proliferation by the growth-factor-producing cells. None of 34 nude or SCID mice developed tumors when injected s.c. with uninfected or neor-virus-infected cells. In contrast, all 30 mice injected with GM-CSF- or G-CSF-producing MG63 cells developed tumors which were G418-resistant and factor-producing. Tumor cell DNA showed a polyclonal retroviral integration pattern indistinguishable from that in the DNA of cells injected into mice. Tumors that formed following injection of a mixture of G418-resistant, GM-CSF-producing cells and cells infected with virus containing only the hygror gene contained hygromycin-resistant cells in the same proportion as was present in the original cell mixture. These data indicate that GM-CSF and G-CSF can support the growth of an osteosarcoma cell line both in vitro and in vivo whether the factor is supplied by autocrine production or from exogenous sources.

A rapid procedure for screening fibroblast packaging cell lines for secretion of selectable retrovirus.

A simple method is described for rapidly screening fibroblast packaging cell line clones for high-titer secretion of retroviruses carrying a selectable marker. Virus-containing supernatants are used to infect FDC-P1 myeloid cells in 24-well tissue culture plates, selection is applied and wells containing live cells (infected by retrovirus and thus expressing the selectable marker) are detected after 5 to 10 days. The number of live cells in each well is proportional to the retroviral titer of the infecting supernatant. The assay is quick to set up and allows simultaneous screening of many samples.

Inhibition of the Moloney murine leukemia virus cycle at a post reverse transcriptional step by the netropsin-intercalating hybrid molecule netropsin-oxazolopyridocarbazole.

In a search for new antiretroviral agents acting at the nucleic acid level, two hybrid molecules composed of a bispyrrolecarboxamide chain related to netropsin, linked to the intercalating chromophore oxazolopyridocarbazole, were tested on the cycle of a defective Moloney murine leukemia virus (M.MuLV) derived from the SVX shuttle and expressing resistance to the G418 antibiotic. The drug netropsin-oxazolopyridocarbazole (Net-OPC), which displays a binding preference to duplex DNA containing A + T bases, inhibits the retroviral replicative cycle (IC50 = 4.8 microM). In contrast, the related molecule (bis)pyrollecarboxamide-oxazolopyridocarbazole (Bpc-OPC) devoid of sequence preference as well as the elemental components of Net-OPC, namely OPC, pentyl-OPC and netropsin, displays no significant action on the viral cycle. The estimation of cytosolic viral DNA in infected cells using quantitative polymerase chain reaction suggests that Net-OPC impairs a post retrotranscriptional step of the viral cycle.

Involvement of the spleen in preleukemic development of a murine retrovirus-induced promonocytic leukemia.

An acute myeloid leukemia can result from the inoculation of Moloney murine leukemia virus into BALB/c mice undergoing a 2,6,10,14-tetramethylpentadecane-induced chronic inflammatory response in the peritoneal cavity. This leukemia is ultimately observed in the peritoneal cavity as an ascites with cells infiltrating the granulomatous tissue. It has been proposed, however, that hematopoietic organs such as the spleen and bone marrow are involved in preleukemic development of Moloney murine leukemia. Therefore, to determine if the spleen plays a role in this development, mice were splenectomized at various times relative to virus inoculation. When splenectomies were performed 3 days before and 2, 4, 6, and 8 weeks after virus inoculation there was, in all cases, a decreased death rate compared to sham-splenectomized controls. The greatest difference in death rate due to promonocytic leukemia was observed when mice were splenectomized at 4 weeks after virus inoculation. The decrease in disease incidence observed as a result of splenectomy was not caused by decreased virus spread in hematopoietic organs or an alteration in the profile of the cellular infiltrate in the granuloma. It was found, however, that the spleens of 2,6,10,14-tetramethylpentadecane-treated mice, relative to those of normal mice, have a significantly increased number of granulocyte-macrophage colony-forming cells and a slightly increased number of multipotential colony-forming cells. These observations suggest that a population of target cells for transformation, consisting of granulocyte-macrophage precursor cells, may reside in the spleen. Alternatively, partially transformed cells may reside temporarily in the spleen during the developmental stages of the disease process.

Differential expression of subspecies of polyomavirus and murine leukemia virus enhancer core binding protein, PEBP2, in various hematopoietic cells.

The core sequence of the enhancer of murine leukemia virus (MuLV) long terminal repeat is highly conserved in a large number of MuLV strains and appears to play an essential role when SL3-3 or Moloney strains induce T cell lymphoma in mice. We found by using the electrophoretic mobility shift assay that a polyomavirus enhancer core-binding protein, PEBP2, bound to this core motif of MuLV. We also noted that PEBP2 in several hematopoietic cell lines derived from B lymphocyte, macrophage and myelocyte lineages migrated significantly faster than the authentic PEBP2 detected in NIH3T3 fibroblasts. Interestingly, PEBP2 detected in the cell lines of T lymphocyte lineage appeared to contain both types, which were indistinguishable in electrophoretic mobility from those of NIH3T3 and of B lymphocyte, macrophage and myelocyte lineages. The treatment of the nuclear extract containing PEBP2 with phosphatase generated PEBP3, which is a subcomponent of PEBP2 and retained the same DNA-binding specificity as PEBP2. The altered mobility of hematopoietic cell-derived or T lymphocyte-derived PEBP2 was found to be due to the alteration of the mobility of PEBP3. Based on the distinct mobility of PEBP2/3 of T lymphocytes from those of other hematopoietic cells, we discuss the implication of PEBP2 in MuLV-induced T cell leukemia and T cell-specific gene expression.

Differences in retention and expression of transfected human cytomegalovirus Towne XbaI-E transforming fragment in human cervical and NIH 3T3 lines.

Human cervical and NIH 3T3 cells were transfected with the XbaI-E-transforming fragment of human cytomegalovirus strain Towne. Southern blot hybridization showed that 3 of 4 transformed NIH 3T3 cell lines retained only the mtrII subfragment of Towne XbaI-E, but not the mtrIII subfragment. Even though mtrII was retained, no viral transcripts were detected. Analysis of genomic DNAs isolated from three independently derived lines of Towne XbaI-E-transfected human exocervical epithelial cells previously immortalized by human papillomavirus type 16 (CX16-2/Towne-E) revealed the retention of both mtrII and mtrIII subfragments of Towne XbaI-E even after greater than 30 subpassages. Southern blot hybridizations indicated the integration and rearrangement of mtrII as well as mtrIII. Poly (A)+RNA analysis of CX16-2/Towne-E line revealed a 1.9-kb transcript which hybridized to mtr III. In contrast, no viral transcript from the mtrII region was detected in these cells. The pattern of HPV-16 DNA sequences and the profile of RNA transcripts were similar in the parental human exocervical cells (CX16-2) and in the CX16-2/Towne-E cells. Thus far, the CX16-2/Towne-E lines are nontumorigenic in nude mice. This study highlights not only differences in the ability of Towne XbaI-E to transform rodent cells and not human cells but also differences in the retention and expression of mtrII and mtrIII in these cells.

Reduction in the calcium requirement for growth is correlated with intracellular calcium stores in normal and SV40-transformed NIH 3T3 cells.

Untransformed NIH 3T3 cells do not proliferate in media with reduced calcium, while SV40-transformed NIH 3T3 cells do. Intracellular calcium stores of untransformed cells were depleted to a higher extent than those of transformed cells under these conditions, which led to a decreased intracellular calcium transient in response to serum, compared to SV40-transformed cells. Furthermore, untransformed cells could be gradually adapted to proliferate in the low-calcium medium and, after adaptation, maintained their stores and serum response in low calcium media. Our experiments indicate that it is the ability of the cells to maintain adequate calcium stores in low calcium media that correlates with a full serum response and the ability to proliferate, rather than any differences reflected in alterations of resting calcium levels.