The limbal niche in the corneoscleral junction of the eye, habitat of the limbal epithelial stem cells (LESC), facilitates corneal epithelial regeneration by providing physical support and chemical signalling. Anatomical structures within the limbus, namely, limbal epithelial crypts and focal stromal projections, are believed to function as a putative niche for LESCs. In this study, the impact of age on the topography of this niche was investigated. Also, the relationship between niche topography and limbal epithelial cell phenotype was assessed. Ex vivo imaging of the limbus in cadaveric tissue of donors aged from infancy to 90 years was carried out using electron and confocal microscopy. The data suggested that the area occupied by the crypts was sharply reduced after the age of 60 years. The niche microstructures also became smoother with donor age. The phenotypic assessment of cultured limbal epithelial cells harvested from donors of different ages showed that the levels of putative stem cell markers as well as telomerase activity and telomere length remained unchanged, regardless of niche topography. However, the colony forming efficiency of the cultures was significantly reduced with age (p < 0.05). This is the first comprehensive study of the effect of age on the structural and phenotypic characteristics of the human limbal niche. The results have a significant biological value as they suggest a correlation of limbal architecture with decline of re-epithelialisation rate in older patients. Overall, the data also suggest that LESCs harvested from younger donors may be more suitable for cultured LESC therapy production.
Stem cells; Cornea; Stem cell markers; Telomerase activity; Telomere length; Imaging
To examine whether human endogenous retrovirus K10 is associated with autoimmune rheumatic disease.
A novel multiplex reverse transcription polymerase chain reaction (RT‐PCR) system was developed to investigate HERV‐K10 mRNA expression in patients with rheumatoid arthritis.
40 patients with rheumatoid arthritis, 17 with osteoarthritis, and 27 healthy individuals were recruited and total RNA was extracted from peripheral blood mononuclear cells (PBMCs) and analysed using multiplex RT‐PCR for the level of HERV‐K10 gag mRNA expression. Southern blot and DNA sequencing confirmed the authenticity of the PCR products.
Using the histidyl tRNA synthetase (HtRNAS) gene as a housekeeping gene in the optimised multiplex RT‐PCR, a significantly higher level of HERV‐K10 gag mRNA expression was found in rheumatoid arthritis than in osteoarthritis (p = 0.01) or in the healthy controls (p = 0.02).
There is enhanced mRNA expression of the HERV‐K10 gag region in rheumatoid arthritis compared with osteoarthritis or healthy controls. This could contribute to the pathogenesis of rheumatoid arthritis.
human endogenous retroviruses; rheumatoid arthritis; peripheral blood mononuclear cells; histidyl tRNA synthetase
Four-coordinate (Pt(II)) platinum-based anticancer drugs are widely used in primary or palliative chemotherapy and produce considerable efficacy in certain clinical applications, for example testicular cancer. However, in many cancers the Pt(II) drugs are beset by poor efficacy mainly due to suboptimal pharmacokinetic properties. Consequently, the six-coordinate (Pt(IV)) class of Pt drugs were developed to improve platinum efficacy by (i) increasing stability, (ii) reducing reactivity, (iii) increasing lipophilicity, and (iv) nuclear targeting. However, comparatively little information is available on the pharmacokinetic properties of these compounds within solid tumour tissue. In the present study, the distribution and fluxes of [14C]-labelled [PtCl2(en)] (where en stands for ethane-1,2-diamine) and cis,trans-[PtCl2(OH)2(en)] drugs were determined in the multicell layer (MCL) tumour model comprising colon cancer cells. Flux data were analysed by mathematical modelling of drug diffusion and cellular uptake in the transport system. The flux of the Pt(IV) compound through the MCL was not significantly different to that of the Pt(II) drug nor were the diffusion coefficient or tissue uptake; the latter confirmed with elemental imaging analysis by synchrotron radiation induced X-ray emission. However, the flux of the Pt(IV) through the MCL was increased by hydrostatic pressure, thereby demonstrating the potential to target cancer cells further away from the vessels with six-coordinate platinum drugs.
cisplatin; convection; diffusion; drug delivery; multicell layer; 3D tumour model
The quiescent cell population of tumours poses a barrier to the success of many cancer therapies. Most chemotherapeutic drugs target proliferating cells, but the growth fraction of many tumours is low. Based on the multicellular tumour spheroid model, a system was developed using human colon adenocarcinoma (DLD-1) cells to mimic the microenvironment of quiescent microregions of solid tumours. The quiescent tumour spheroids (TSQ) showed decreased expression of the proliferation marker Ki-67 and increased expression of the quiescence marker p27kip1 compared to proliferating spheroids (TSP). The quiescent status of the TSQ was confirmed by long-term growth assessment. The quiescence was completely reversible demonstrating that the TSQ retained the ability to proliferate and morphological assessment by light microscopy confirmed the absence of significant apoptosis. When the efficacy of widely used chemotherapeutic drugs was determined, vinblastine, doxorubicin, cisplatin and 5-fluorouracil (5-FU) all produced significant cell death in the TSP. However, while still effective, the potencies of doxorubicin and cisplatin were significantly reduced in TSQ. In contrast, 5-FU and vinblastine did not produce cell death in the TSQ. In summary, TSQ show considerable resistance to a panel of established chemotherapeutic agents and represent a useful model for evaluating the efficacy of drugs and other cancer therapies in quiescent tumours.
quiescent; tumour; spheroid; microenvironment; chemotherapy; multicellular resistance
drug distribution; P-glycoprotein; tumour models; confocal fluorescence microscopy; drug resistance
The gene encoding the multidrug resistance P-glycoprotein (P-gp) is duplicated in rodent species and the functional basis for this remains unresolved. Despite a high sequence similarity, the mouse P-gp1a and P-gp1b isoforms show distinct patterns of tissue distribution which suggest a specific role of the P-gp1b isoform in steroid transport. In the present study possible biochemical differences between the isoforms were directly investigated at the level of drug interaction. There was no detectable difference in the affinity or binding capacity of the two isoforms towards [3H]vinblastine at equilibrium. Similarly, the rate at which [3H]vinblastine associates with P-gp was indistinguishable between the two isoforms. Some modest differences were observed in the relative abilities of the multidrug-resistant (MDR) reversing agents CP100-356, nicardipine and verapamil to displace equilibrium [3H]vinblastine binding to P-gp1a and P-gp1b. The steroid hormone progesterone displayed a low affinity (Ki = 1.2 ± 0.2 μM for P-gp1a and 3.5 ± 0.5 μM for P-gp1b), suggesting an unlikely role as a physiological substrate. Thus the mouse isoforms do not appear to exhibit functional differences at the level of initial substrate interaction with protein. © 1999 Cancer Research Campaign
P-glycoprotein; MDR; drug binding; steroid hormones
XR9051 (N-(4-(2-(6,7-Dimethoxy-1,2,3,4-tetrahydro-2-isoquinolyl)ethyl)phe nyl)-3-((3Z,6Z)-6-benzylidene-1-methyl-2,5-dioxo-3-pipera zinylidene) methylbenzamide) was identified as a potent modulator of P-glycoprotein-mediated multidrug resistance (MDR) following a synthetic chemistry programme based on a natural product lead compound. The activity of XR9051 was determined using a panel of human and murine drug-resistant cell lines (H69/LX4, 2780AD, EMT6/AR 1.0, MC26 and P388/DX Johnson). XR9051 was able to reverse resistance to a variety of cytotoxic drugs, including doxorubicin, etoposide and vincristine, which are associated with classical MDR. At a concentration of 0.3-0.5 microM, XR9051 was able to fully sensitize resistant cells to cytotoxics, whereas little or no effect was observed on the corresponding parental cell lines. No effect of XR9051 was observed on the response of cells to non-MDR cytotoxics such as methotrexate and 5-fluorouracil. XR9051 was consistently more potent than cyclosporin A (CsA) and verapamil (Vpm) in all assays used. XR9051 inhibited the efflux of [3H]daunorubicin from preloaded cells and, unlike CsA and Vpm, remained active for several hours after removal of resistance-modifying agent. In photoaffinity labelling experiments employing [3H]azidopine, XR9051 was able to displace binding to P-glycoprotein. In binding studies using [3H]vinblastine, XR9051 was shown to be a potent inhibitor of the binding of the cytotoxic to P-glycoprotein (EC50 = 1.4 +/- 0.5 nM). Taken together, the results indicate that XR9051 reverses the MDR phenotype through direct interaction with P-glycoprotein.
Staphylococcus aureus corneal infection results in extensive inflammation and tissue damage. Our previous studies of bacterial mutants have demonstrated a role for alpha-toxin in corneal virulence. This study analyzes, by genetic rescue experiments, the virulence of mutants affecting alpha-toxin and beta-toxin activity and demonstrates the ocular toxicity of these purified staphylococcal proteins. Three types of isogenic mutants were analyzed: (i) mutants specifically deficient in alpha-toxin (Hla) or beta-toxin (Hlb), (ii) a mutant deficient in both Hla and Hlb, and (iii) a regulatory mutant, deficient in the accessory gene regulator (agr), that produces reduced quantities of multiple exoproteins, including alpha- and beta-toxins. Plasmids coding for Hla and Hlb (pDU1212 and pCU1hlb, respectively) were used to restore toxin activity to mutants specifically deficient in each of these toxins. Either corneas were injected intrastromally with logarithmic-phase S. aureus or purified alpha- or beta-toxins were administered to normal eyes. Ocular pathology was evaluated by slit lamp examination and myeloperoxidase activity of infiltrating polymorphonuclear leukocytes. Corneal homogenates were cultured to determine the CFU per cornea. Eyes infected with the wild-type strain developed significantly greater corneal damage than eyes infected with Agr-, Hlb-, or Hla- strains. Epithelial erosions produced by parent strains were not produced by Agr- or Hla- strains. Hlb+ strains, unlike Hlb- strains, caused scleral edema. Plasmid pDU1212 restored corneal virulence to strain DU1090 (Hla-), and plasmid pCU1hlb restored corneal virulence to strain DU5719 (Hlb-). Application of purified alpha-toxin produced corneal epithelial erosions and iritis, while application of beta-toxin caused scleral inflammation. These studies confirm the role of alpha-toxin as a major virulence factor during S. aureus keratitis and implicate beta-toxin, a mediator of edema, as a lesser contributor to ocular damage.
In this investigation we determined the dynamics of herpes simplex virus type 1 (HSV-1) DNA and latency-associated transcripts (LAT) in the latently infected rabbit trigeminal ganglion. Rabbit eyes were infected with either the McKrae strain or the l7Syn+ strain of HSV-1. Rabbits were sacrificed between 5 and 360 days after infection and their trigeminal ganglia were analyzed for the number of HSV DNA genomes and the number of neuronal cells expressing LAT. There was no statistically significant change in the number of HSV genomes or the number of neuronal cells expressing LAT in these ganglia between 20 and 360 days after infection. For both strains, the amount of HSV DNA averaged 16.8 genomes per 100 cells, and 9.2% of the neurons expressed LAT. There were 17 to 34 HSV genomes per LAT-expressing neuronal cell. The number of LAT-expressing neurons did not change over the 360 days. Spontaneous reactivation (HSV-1 recovery in tear film) and recurrence (HSV-1-specific epithelial lesions) occurred during the period of this study; however, these events did not alter the quantity of HSV-1 DNA or the number of LAT-expressing cells. These results suggest that after the latent infection is established, the viral DNA in the ganglia does not replicate to any measurable extent over long periods of latency, since no significant change in the number of HSV genomes occurs. The results also suggest that only a very small number of latently infected neuronal cells are needed to produce infectious HSV-1 during reactivation.
Tamoxifen is an anti-oestrogen which is currently being assessed as a prophylactic for women at high risk of breast cancer. Taxoxifen has also been shown to reverse multidrug resistance in P-glycoprotein (P-gp)-expressing cells, although the mechanism of action is unknown. In this study we demonstrate that tamoxifen interacts directly with P-gp. Plasma membranes from P-gp-expressing cells bound [3H]tamoxifen in a specific and saturable fashion. A 180 kDa membrane protein in these membranes, labelled by the affinity analogue tamoxifen aziridine and azidopine, was shown to be P-gp. Tamoxifen reduced the binding of vinblastine and azidopine to P-gp, and tamoxifen increased [3H]vinblastine accumulation in P-gp-expressing cells to levels approaching those in non-P-gp-expressing cells. However, the cellular accumulation of [3H]tamoxifen itself was not influenced by the presence of P-gp. Thus, tamoxifen appears to reverse multidrug resistance by binding to P-gp and inhibiting the transport of cytotoxic drugs, but does not itself appear to be transported by the protein.
Staphylococcus aureus produces a variety of proteins, including alpha-toxin and protein A, that could contribute to corneal tissue damage during keratitis. We examined corneal infections produced by intrastromal injection of four S. aureus strains--three isogenic mutants, one lacking alpha-toxin (Hly- Spa+), one lacking protein A (Hly+ Spa-), and one lacking both alpha-toxin and protein A (Hly- Spa-), and the wild type (Hly+ Spa+)--in a rabbit model of experimental keratitis. Rabbit corneas were injected intrastromally with 100 CFU of one of the four strains, and the eyes were examined by slit lamp biomicroscopy over a 25-h period. Corneal homogenates were used for determination of CFU and neutrophil myeloperoxidase activity at 5-h intervals. All strains had the same logarithmic growth curve from 0 to 10 h postinfection, after which CFU remained constant at 10(7) CFU per cornea. By 15 h postinfection, slit lamp examination scores were significantly higher for eyes infected with Hly+ strains than for Hly(-)-infected eyes. At this time, distinct epithelial erosions were seen in Hly(+)-infected eyes but not in Hly(-)-infected eyes. Myeloperoxidase activity was significantly greater for Hly(+)-infected corneas than for Hly(-)-infected corneas at both 20 and 25 h postinfection. Spa(+)- and Spa(-)-infected eyes showed no differences in slit lamp examination scores or myeloperoxidase activities. These results suggest that alpha-toxin, but not protein A, is a major virulence factor in staphylococcal keratitis, mediating the destruction of corneal tissue in eyes infected with this bacterial pathogen.
This study was conducted to determine whether the age of the host influences the pathogenesis and therapeutic outcome of drug-treated Pseudomonas aeruginosa keratitis. Young (3- to 5-month-old) and old (1.5- to 3-year-old) rabbits were intrastromally infected with P. aeruginosa ATCC 27853. Sixteen hours later, rabbits in both age subpopulations were divided into three groups and treated topically as follows: group 1, phosphate-buffered saline; group 2, 0.3% ciprofloxacin; and group 3, 0.3% ciprofloxacin, 1.0% prednisolone, and 0.03% flurbiprofen. Drops were given every 15 min for 1 h and then every 30 min for 9 h. At 27 h postinfection, ocular pathology was graded with a slit lamp examination (SLE) scoring system. Aqueous humor was collected for ciprofloxacin quantitation, and corneas were harvested for bacterial enumeration and estimation of polymorphonuclear leukocytes. Young rabbits had more severe inflammation and pathology than old rabbits. At 27 h postinfection, SLE scores and polymorphonuclear leukocyte numbers were significantly higher for young rabbits than old rabbits (P < 0.02), regardless of treatment. Prednisolone and flurbiprofen significantly reduced SLE scores in both age groups (P < 0.03) without affecting the antimicrobial efficacy of ciprofloxacin.
Cell lines selected (CHRC5) and transfected (LR-73-1A) for multi-drug resistance have total lipid compositions which are indistinguishable between resistant and parental cells. Lipid composition was evaluated by 1H NMR and the total fatty acid content by GLC. No change in surface hydrophobicity, as measured with the fluorescent probe dansyl-PE, was observed as a result of transfection of CHO cells with the mdr1 gene. However, the selected cell line, CHRC5, showed a decreased surface hydrophobicity. This decreased surface hydrophobicity was indicated by an 8 nm increase in the fluorescence emission of dansyl-PE in the CHRC5 cell line compared with the AB1. Both resistant cell lines showed an increase in the polarisation of the fluorescent probe, TMA-DPH in the plasma membranes corresponding to a 14% and a 24% change in fluorescence polarisation for the selected and transfected cell lines, respectively. This is indicative of reduced mobility of the acyl chains in the resistant cell lines. Both the CHRC5 and the transfected cell lines showed almost a 2-fold increase in the initial rate of membrane cycling. The membrane cycling could be inhibited by a known bilayer stabiliser, the N-carbobenzoxy-D-Phe-L-Phe-Gly. These results indicate that the properties of the plasma membrane from resistant cells are altered compared with their parental cell line.
A variety of chemically defined compounds were tested to characterize the substrate specificity of the influenza C virus esterase and to determine whether a substrate could be found that would be useful in an assay to detect the virus. Two new substrates, alpha-naphthyl acetate and alpha-naphthyl propionate, were identified; alpha-naphthyl acetate was employed to develop an assay specific for influenza type C virus in MDCK cells. The assay was sufficiently sensitive to detect esterase activity in a single cell and distinguished influenza C virus infections from those of types A and B viruses. Infected cells could be detected as early as 8 h postinfection, with maximal enzyme detection occurring at 24 h. Assay of influenza C virus in the chorioallantoic or amniotic fluid of infected eggs was performed by applying fluids directly onto nitrocellulose strips and then incubating with alpha-naphthyl acetate. Both the cellular and nitrocellulose-bound assays are rapid, inexpensive, and easy to perform, offering advantages for use in clinical laboratories.
Pseudomonas aeruginosa keratitis was induced in rabbits to study the effects of corneal infection on the delivery of tobramycin by iontophoresis. Some rabbits were treated by use of an eye cup with no current as a control for iontophoresis, and others were treated with fortified drops (1.36%) delivered topically for comparison with results of earlier studies. One hour after treatment with tobramycin, the concentration of drug in the infected corneas was compared with that achieved in mock-infected and uninfected eyes. Iontophoresis of 25 mg of tobramycin per ml at 0.8 mA for 10 min delivered significantly more drug (P = 0.0001) to corneal tissue than did drops or use of an eye cup without current in P. aeruginosa-infected eyes mock-infected eyes, or uninfected eyes. Tobramycin concentrations in the infected corneas (605.9 micrograms/g) were not significantly different (P = 0.815) from the concentrations in mock-infected eyes (641.4 micrograms/g), but were lower (P = 0.007) than those obtained by iontophoresis in uninfected corneas (853.6 micrograms/g). Use of an eye cup without current delivered tobramycin equally to infected, mock-infected, and normal eyes, i.e., 176.5, 171.0, and 163.1 micrograms/g, respectively (P greater than 0.709). Tobramycin delivered by use of fortified drops delivered topically was detectable in mock-infected corneas (20 micrograms/g) and P. aeruginosa-infected corneas (6.0 micrograms/g). These results suggest that iontophoresis has value as an ocular drug delivery system and that an eye cup could also be useful in a therapeutic regimen for ocular infections.
A colistin-resistant mutant of Klebsiella pneumoniae served well as a donor but not as a recipient in conjugation. A nearly 1,000-fold difference between colistin-susceptible and colistin-resistant forms of this strain was observed by using donors of plasmids of four incompatibility groups. Recipient efficiency was not restored by filter matings.
Influenza C virus contains a hemagglutinin and a receptor-destroying enzyme (RDE) whose specificities remain undetermined. In rat serum, there is a molecule that binds specifically to C virus, inhibiting its hemagglutinin. The complex between C virus and the rat serum inhibitor (RSI) was determined to be stable at 4 degrees C, but was disrupted within 20 to 90 min at 23 or 37 degrees C. Virus emerged from the complex with numerous functions intact, whereas the RSI at this point was inactivated, i.e., incapable of further inhibitory reactions with C virus. RSI could not be inactivated at these temperatures by nonviral components of allantoic fluid of infected chicken embryos; however, RSI inactivation was achieved by preparations of sucrose gradient-purified virus. Neutralization of viral hemagglutination activity with antiviral antibody protected the RSI from inactivation. RSI inactivation occurred at temperatures at which the viral RDE was active, and inhibition of viral RDE by periodate treatment sharply reduced the ability of virus to inactivate the RSI. One interpretation of the data suggests that RSI is a receptor analog reactive with both the hemagglutinin and RDE of C virus and that RSI inactivation is an assay of influenza C viral RDE.
Sera from persons of four age groups (1 to 2 years, 2 to 5 years, 20 to 30 years, and 65 to 85 years) were analyzed for hemagglutination inhibition (HI) activity for influenza C virus. Significant HI activity was found in 66% of the 237 sera tested, and titers ranged from 8 to 512. In the yoiung adult group, 96% had antibody and the highest mean titer (74.7) of any age group. Positive sera were far less common in young children (36 to 47%), and relatively low titers (18.3) were common among adults over 65. The high percentage of sera with antibody to influenza C virus suggests that infections with this virus occur at a rate greater than previously recognized. The high percentage of young adults with elevated levels of HI antibody suggested either that an immune response to influenza C infections is common or that the observed HI activity might be attributable, in part at least, to nonspecific inhibitors in the sera. We showed both directly and indirectly that most if not all the inhibitory activity in the human sera we examined was due to specific antibody, mostly immunoglobulin G. This conclusion is based on the finding that the single serum protein fraction with HI activity was found to have a molecular weight equivalent to that of 7S antibody (150,000) and that the HI activity was removed by absorption to staphyloccal protein A. Moreover, immunoglobulin from only HI-positive sera bound specifically to cells infected with influenza C virus, as shown by inhibition of hemadsorption and immunofluorescence. These findings were supported by similar results obtained with chicken antisera to C virus.
Kanamycin resistance in eight strains of Klebsiella pneumoniae isolated during an outbreak of infection in a neonatal intensive care unit was found to be transferable and mediated by neomycin phosphotransferase. After gentamicin was used to control infections caused by kanamycin-resistant organisms, a strain resistant to gentamicin emerged. Gentamicin resistance in this ninth strain was not transferable and was accompanied by resistance to tobramycin, amikacin, and streptomycin. Enzymatic modifications of aminoglycosides other than neomycin and kanamycin could not be demonstrated either by filter binding assays or by electrophoresis of a radioactive aminoglycoside substrate. The strain with broad aminoglycoside resistance contained six plasmid deoxyribonucleic acid bands, none of which appeared to be different in molecular weight from plasmid deoxyribonucleic acid bands in strains isolated before the institution of gentamicin therapy. The broadening of resistance was accompanied by reduced uptake of radioactively labeled streptomycin and gentamicin. The relationship between aminoglycoside resistance and reduced drug transport in the absence of any enzymatic modification is discussed.
Influenza C virus was propagated successfully in primary chicken embryo lung (CEL) and fibroblast cells and in Madin-Darby canine kidney (MDCK) cells. In other cell lines, either no virus or only noninfectious hemagglutinin (HA) was produced. In productively infected cells (CEL), HA and infectious virus appeared by 24 h and reached a maximum by 36 to 48 h, cell-associated virus remaining at a constant low level. Infected Vero cells produced noninfective HA by 24 h which also remained predominantly cell associated until 60 to 72 h, when the cells disintegrated. Viral antigen was demonstrable on membranes of both CEL- and Vero-infected cells at 24 h; Vero cells yielded membrane vesicles containing HA, but none of the spherical or filamentous viral particles synthesized in CEL cells. Influenza C virus produced in cell culture or in eggs differed in several important respects from A and B viruses and from Newcastle diseases virus. All influenza C preparations, regardless of infectivity or source, lacked detectable neuraminidase activity, yet retained the ability specifically to inactivate receptors only for influenza C. Influenza C HA was not inhibited by soluble glycoproteins highly active against HA of A virus. A rat serum glycoprotein uniquely inhibited influenza C by binding to the surface components of virious.
The arsenate poisoning of R17 phage eclipse in Escherichia coli cultures grown in glycerol-containing medium has been found to be mediated by a dramatic loss in cell-associated F pili. Poisoning was very rapid and was nearly complete within 3 min at 37 C. The loss of pili was reflected by a 90% reduction in the ability of these cells to attach ribonucleic acid phage and by a reduction in the pili-per-cell ratio from 1.36 to 0.04 as determined by electron microscopy. Neither the integrity of the pilus per se nor the attachment process was affected by arsenate, for cell-free pili treated with arsenate retained the ability to attach phage. The arsenate effect on F piliation could be reversed readily without any loss in cell viability. This restoration of pili occurred under conditions of inhibited protein synthesis, implying that pools of pili protein exist in cells. In contrast to this phenomenon, cells grown in glucose-containing media were mainly resistant to arsenate poisoning as determined by phage attachment and the number of pili per cell. These results implied that arsenate poisons the ability of cells to synthesize and maintain F pili under certain specific conditions. The possible mechanism of this poisoning is discussed.