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Biomedical subjects

K Gustafsson

Publications and source records attributed to K Gustafsson.

At least 37 records · Page 2Linked to original sources
PubMed2000

Specific suppression of interleukin 2 biosynthesis by synthetic antisense oligodeoxynucleotides does not influence allograft rejection.

BACKGROUND: Interleukin (IL)-2 supplementation can reverse both blood transfusion-induced tolerance to kidney allografts and spontaneous tolerance to liver allografts in rats. Moreover, IL-2 expression is frequently suppressed in models of allograft tolerance. The failure of IL-2 biosynthesis might therefore play a critical role in tolerance induction. METHODS: Three antisense oligodeoxynucleotides (AS-1, AS-2, AS-3) to rat IL-2, and a control oligo (C-1) consisting of a scrambled version of AS-1, were evaluated for gene-specific suppression of IL-2 biosynthesis in vitro and in vivo, and for their effects on kidney allograft survival. Reverse transcriptase-polymerase chain reaction and IL-2 protein assays were used to assay concanavalin A-driven IL-2 biosynthesis by lymph node lymphocytes in vitro. PVG recipients of Dark Agouti kidney allografts were treated with the oligos. Graft survival and IL-2 biosynthesis by reverse transcriptase-polymerase chain reaction in spleen and graft biopsy specimens were assessed. RESULTS: The AS-1 oligo, but not the AS-2, AS-3 or C-1 oligos, suppressed concanavalin A-driven IL-2 biosynthesis for the 4 days of culture. This effect was dependent on delivery of the AS-1 oligo with lipofectamine. Supplementation with exogenous IL-2 reversed the suppression of lymphocyte proliferation in AS-1-treated cultures. Administration of AS-1 intravenously at 10 mg/kg/day to PVG recipients of Dark Agouti kidney allografts suppressed IL-2 (but not IL-6, interferon-gamma, or tumor necrosis factor-alpha) synthesis in the grafts of seven of nine rats, as measured in biopsy specimens taken at days 2-7. By contrast, all nine control grafts strongly expressed IL-2. However, neither graft histopathology nor graft survival was affected. CONCLUSIONS: Antisense oligonucleotides can powerfully suppress IL-2 biosynthesis in vitro and in allograft recipients in vivo, but this does not affect kidney allograft rejection.

Animals↗
PubMed2000

Measurement of tensor polarization in elastic electron-deuteron scattering at large momentum transfer

Tensor polarization observables ( t(20), t(21), and t(22)) have been measured in elastic electron-deuteron scattering for six values of momentum transfer between 0.66 and 1.7 (GeV/c)(2). The experiment was performed at the Jefferson Laboratory in Hall C using the electron High Momentum Spectrometer, a specially designed deuteron magnetic channel and the recoil deuteron polarimeter POLDER. The new data determine to much larger Q2 the deuteron charge form factors G(C) and G(Q). They are in good agreement with relativistic calculations and disagree with perturbative QCD predictions.

Journal Article↗
PubMed2000

Tissue-binding properties of a synthetic peptide DNA vector targeted to cell membrane integrins: a possible universal nonviral vector for organ and tissue transplantation.

BACKGROUND: Gene delivery through a nonviral, receptor-mediated system widely expressed in transplanted tissue would have important advantages in transplantation, where gene delivery is performed ex vivo. Integrins are widely expressed cell surface receptors and can be targeted for gene delivery. METHODS: A synthetic 31 amino acid DNA vector (polylysine-molossin) comprising a 15-amino acid moiety for targeting cellular integrins (derived from the snake venom, molossin) and a 16-amino acid polylysine moiety for DNA-binding, has been evaluated. The 31-amino acid vector, as well as its separate 15-amino acid integrin-binding and (lys)16 components, were individually synthesized, and a monoclonal antibody was raised to the molossin peptide for these studies. Binding to cell lines and tissue sections and capacity for gene delivery were examined. RESULTS: Flow cytometric studies with the ECV304 cell line demonstrated that the binding of polylysine-molossin and polylysine-molossin/DNA complexes involved both electrostatic and integrin-mediated interactions with the cells, with the electrostatic binding being sufficient for maximal binding. However, binding to cellular integrins was essential for successful gene transfer. Binding studies on frozen tissue sections of the rat and pig demonstrated that the molossin peptide bound to many cell types of interest in transplantation, but not to all. Among the negative tissues were vascular endothelium and pancreatic islets. Small species differences in tissue binding were noted between the rat and pig. CONCLUSIONS: This study defines the cooperative nature of the binding of this vector system to target cells and establishes the cell types most likely to be effectively targeted for DNA transfer.

Antineoplastic Agents↗
PubMed2000

In vitro investigation of factors important for the delivery of an integrin-targeted nonviral DNA vector in organ transplantation.

BACKGROUND: Polylysine-molossin is a 31 amino acid synthetic peptide that has previously been demonstrated to function as a DNA vector in vitro for cell lines and for the cornea. It incorporates the 15 amino acid integrin-binding domain of the venom of the American pit viper, Crotalus molossus molossus as the targeting moiety and a chain of 16 lysines as the DNA-binding moiety. The objective of this study was to evaluate several parameters of importance for in vivo applications. METHODS: Binding and tissue distribution of the vector/DNA complexes were followed by a monoclonal antibody to the vector, or by the use of fluorescein-labeled DNA. Standard in vitro transfections were used to monitor effective gene transfer. RESULTS: (1) Optimal DNA/vector concentration. Saturation of vector/DNA binding sites on the ECV304 cell line occurred at 6 microg/ml of DNA. The concentration of vector/DNA complexes required for optimal gene transfection was found to be 2-8 microg/ml of DNA, corresponding to the concentration needed for saturation binding. (2) Optimal target cell exposure time. Vector/ DNA complexes saturated target cell binding sites within 5 min of incubation. However, lengthy exposure times (>2-3 hr) to the transfection medium were essential for substantial gene transfer. This was a consequence of two complementary factors. First, it was important that target cells be exposed to vector/DNA complexes for approximately 1 hr at 37 degrees C. Saturation of target sites at 4 degrees C and then removal of the transfection medium was much less effective. Second, exposure to chloroquine for 8-10 hr after uptake of vector/DNA complexes was essential for optimal gene transfer. (3) Inhibitory effects of serum. Exposure of complexes to even 1% serum before transfection, markedly inhibited gene transfer. However, target cells previously saturated with vector/DNA complexes and then exposed to 10% serum showed substantial gene transfer. (4) Extravasation and binding stability in vivo. Cold ex vivo perfusion of rat hearts with vector/DNA complexes demonstrated that little, if any, complex moved out of the vascular system. After transplantation of the heart, most of the complex bound to the vasculature was lost within 30 min of reestablishing the blood circulation. CONCLUSIONS: Careful attention to several parameters of little importance in vitro need to be paid for optimal in vivo application of DNA vector systems.

Cold Temperature↗
PubMed2000

Constitutive production of interleukin-1alpha mRNA and protein in the developing rat testis.

Interleukin-1 (IL-1), a multifunctional cytokine produced mainly by activated macrophages, is also produced in the intact testis. Rat testicular IL-1 was found to be identical to IL-1alpha, judged by immunoneutralization of the bioactive protein and sequence comparison of cloned rat testicular and macrophage pro-IL-1alpha cDNA. Testicular IL-1alpha mRNA was first demonstrated on postnatal day 15, and the corresponding bioactive protein from day 20. IL-1alpha mRNA was still low on day 20, but then increased rapidly in parallel with the bioactive protein to establish a plateau level from day 25. In adult testes, IL-1alpha mRNA and immunoreactive protein were low in stage VII of the seminiferous epithelial cycle, whereas other stages showed a clearly detectable expression. In the adult testis, the concentration of IL-1alpha was 75 pg/mg testicular protein (approximately 200 pM). In conclusion, production of testicular IL-1alpha is developmentally and stage-dependently regulated, probably at the transcriptional level, emphasizing an important paracrine role in testicular function.

Animals↗
PubMed2000

Perceived fatigue after mental work: an experimental evaluation of a fatigue inventory.

The aim of this study was to investigate the perceived fatigue after mental work and to test the Swedish Occupational Fatigue Inventory (SOFI). Twenty male and 20 female participants worked with proof reading (2 x 90 min) and a vigilance task (2 x 60 min). After each task session, perceived fatigue was rated with the SOFI and Borg's CR10O-scale. In addition, physiological reactions were registered; blood pressure, heart rate, heart rate variability and muscle activity in corrugator supercilii, as well as measures of performance: reaction time, number of pages read and number of proof errors found, number of detected signals. As expected, the highest ratings were obtained on Lack of energy, Lack of motivation and Sleepiness, particularly after the vigilance task. High ratings after both work tasks were also found on the CR10-scale. Men and women did not differ significantly with respect to their ratings. No clear-cut physiological reactions were found to correlate with ratings of fatigue. The results indicate the validity of the mental dimension of the SOFI.

Electromyography↗
PubMed2000

Sick but yet at work. An empirical study of sickness presenteeism.

STUDY OBJECTIVE: The study is an empirical investigation of sickness presenteeism in relation to occupation, irreplaceability, ill health, sickness absenteeism, personal income, and slimmed down organisation. DESIGN: Cross sectional design. SETTING: Swedish workforce. PARTICIPANTS: The study group comprised a stratified subsample of 3801 employed persons working at the time of the survey, interviewed by telephone in conjunction with Statistics Sweden's labour market surveys of August and September 1997. The response rate was 87 per cent. MAIN RESULTS: A third of the persons in the total material reported that they had gone to work two or more times during the preceding year despite the feeling that, in the light of their perceived state of health, they should have taken sick leave. The highest presenteeism is largely to be found in the care and welfare and education sectors (nursing and midwifery professionals, registered nurses, nursing home aides, compulsory school teachers and preschool/primary educationalists. All these groups work in sectors that have faced personnel cutbacks during the 1990s). The risk ratio (odds ratio (OR)) for sickness presenteeism in the group that has to re-do work remaining after a period of absence through sickness is 2.29 (95% CI 1.79, 2.93). High proportions of persons with upper back/neck pain and fatigue/slightly depressed are among those with high presenteeism (p< 0.001). Occupational groups with high sickness presenteeism show high sickness absenteeism (rho = 0. 38; p<.01) and the hypothesis on level of pay and sickness presenteeism is also supported (rho = -0.22; p<0.01). CONCLUSIONS: Members of occupational groups whose everyday tasks are to provide care or welfare services, or teach or instruct, have a substantially increased risk of being at work when sick. The link between difficulties in replacement or finding a stand in and sickness presenteeism is confirmed by study results. The categories with high sickness presenteeism experience symptoms more often than those without presenteeism. The most common combination is low monthly income, high sickness absenteeism and high sickness presenteeism.

Absenteeism↗
PubMed1999

In vivo modulation of alternative pathways of P-450-catalyzed cyclophosphamide metabolism: impact on pharmacokinetics and antitumor activity.

The widely used anticancer prodrug cyclophosphamide (CPA) is activated in liver by a 4-hydroxylation reaction primarily catalyzed by cytochrome P-4502B and P-4502C enzymes. An alternative metabolic pathway involves CPA N-dechloroethylation to yield chloroacetaldehyde (CA), a P-4503A-catalyzed deactivation/neurotoxication reaction. The in vivo modulation of these alternative, competing pathways of P-450 metabolism was investigated in pharmacokinetic studies carried out in the rat model. Peak plasma concentrations (Cmax) for 4-OH-CPA and CA were increased by 3- to 4-fold, and apparent plasma half-lives of both metabolites were correspondingly shortened in rats pretreated with phenobarbital (PB), an inducer of P-4502B and P-4503A enzymes. However, PB had no net impact on the extent of drug activation or its partitioning between these alternative metabolic pathways, as judged from AUC values (area-under-the-plasma concentration x time curve) for 4-OH-CPA and CA. The P-4503A inhibitor troleandomycin (TAO) decreased plasma Cmax and AUC of CA (80-85% decrease) without changing the Cmax or AUC of 4-OH-CPA in uninduced rats. In PB-induced rats, TAO decreased AUCCA by 73%, whereas it increased AUC4-OH-CPA by 93%. TAO thus selectively suppresses CPA N-dechloroethylation, thereby increasing the availability of drug for P-450 activation via 4-hydroxylation. By contrast, dexamethasone, a P-4503A inducer and antiemetic widely used in patients with cancer, stimulated large, undesirable increases in the Cmax and AUC of CA (8- and 4-fold, respectively) while reducing the AUC of the 4-hydroxylation pathway by approximately 60%. Tumor excision/in vitro colony formation and tumor growth delay assays using an in vivo 9L gliosarcoma solid tumor model revealed that TAO suppression of CPA N-dechloroethylation could be achieved without compromising the antitumor effect of CPA. The combination of PB with TAO did not, however, enhance the antitumor activity of CPA, despite the approximately 2-fold increase in AUC4-OH-CPA, suggesting that other PB-inducible activities, such as aldehyde dehydrogenase, may counter this increase through enhanced deactivation of the 4-hydroxy metabolite. Together, these studies demonstrate that the P-4503A inhibitor TAO can be used to effectively modulate CPA metabolism and pharmacokinetics in vivo in a manner that decreases the formation of toxic metabolites that do not contribute to antitumor activity.

Animals↗
PubMed1999

Isocitrate as calcium ion activity buffer in coagulation assays.

BACKGROUND: Ca(2+) activity close to the physiological concentration of 1.3 mmol/L is essential in blood coagulation. Is this also true for the performance of global diagnostic coagulation assays? We searched for compounds that would buffer Ca(2+) activity at approximately 1.3 mmol/L without disturbing coagulation reactions and investigated whether such Ca(2+) buffering improves diagnostic efficacy in global diagnostic coagulation tests. METHODS: Buffering was investigated by mixing CaCl(2) and 11 candidate compounds and determining Ca(2+) activity. The best candidates were added to mixtures of plasma and thromboplastin to detect interference with coagulation reactions. The best of these candidates, isocitrate, was used to modify an activated partial thromboplastin time (APTT), buffering final Ca(2+) activity to approximately 1.3 mmol/L. Plasma samples from 22 healthy individuals and 120 patients were analyzed with original and modified APTT to determine whether diagnostic efficacy was improved. RESULTS: Two suitable Ca(2+) buffers, citrate and isocitrate, were found. Isocitrate was preferred as being less coagulation inhibitory, a better Ca(2+) buffer, and possibly a better anticoagulant. The isocitrate-modified APTT showed a final Ca(2+) activity of 1.60 +/- 0.07 mmol/L, compared with 2.73 +/- 0.20 mmol/L for the original APTT. The means and SDs for the healthy individuals were determined for both procedures, and the values were used to express patient deviation from normality (difference from mean divided by SD). The deviation was greater for the modified APTT; 4.3 +/- 5.7, compared with 3.6 +/- 5.0 (P <0.005) for the original APTT. CONCLUSIONS: Isocitrate can be used to buffer Ca(2+) activity at physiological concentrations and can serve as an anticoagulant. APTT with isocitrate-buffered Ca(2+) activity shows signs of improved diagnostic efficacy.

Blood Chemical Analysis↗
PubMed1998

Suppression of human anti-porcine T-cell immune responses by major histocompatibility complex class II transactivator constructs lacking the amino terminal domain.

BACKGROUND: The class II transactivator (CIITA) is a bi- or multifunctional domain protein that acts as a transcriptional activator and plays a critical role in the expression of MHC class II genes. We have previously demonstrated that a mutated form of the human CIITA gene, coding for a protein lacking the amino terminal 151 amino acids, acts as a potent dominant-negative suppressor of HLA class II expression. Porcine MHC class II antigens are potent stimulators of direct T-cell recognition by human CD4+ T cells and are, therefore, likely to play an important role in the rejection responses to transgenic pig donors in clinical xenotransplantation. We were, therefore, interested in examining mutated CIITA constructs for their effect on porcine MHC class II expression. METHODS: Stable transfectants of the porcine vascular endothelial cell line PIEC with mutated CIITA constructs were tested for SLA-DR and SLA-DQ induction by recombinant porcine interferon-gamma. Transient transfectants of the porcine B-cell line L23 with the mutated CIITA constructs were tested for the suppression of constitutive SLA-DR and SLA-DQ expression. T-cell proliferation studies were performed using highly purified human CD4+ T cells. RESULTS: In preliminary studies, we demonstrated that transfection of the PIEC line with full-length human CIITA constructs resulted in strong expression of SLA-DR and SLA-DQ antigens, thus establishing the cross-species effectiveness of human CIITA in the pig. The mutated human CIITA constructs were, therefore, tested in the pig. PIEC clones stably transfected with one of these constructs showed up to 99% suppression of SLA-DR and SLA-DQ antigen induction and marked suppression of SLA-DRA mRNA induction. Moreover, transient transfection of the porcine B-cell line L23 showed up to 90% suppression of constitutive SLA-DR and SLA-DQ antigen expression in 5-8 days. In functional studies, interferon-gamma-stimulated PIEC clones transfected with this mutated CIITA construct failed to stimulate purified human CD4+ T lymphocytes. CONCLUSION: Mutated human CIITA constructs are potent suppressors of porcine MHC class II expression.

Amino Acid Sequence↗
PubMed1998

Modulation of P450-dependent ifosfamide pharmacokinetics: a better understanding of drug activation in vivo.

The anti-cancer prodrug ifosfamide (IF) is metabolized by liver P450 enzymes by two alternative pathways. IF is activated to 4-hydroxy IF (4-OH-IF), which ultimately yields the alkylating mustard isophosphoramide, whereas IF N-dechlororethylation inactivates the drug and produces the neurotoxic metabolite chloroacetaldehyde (CA). Both reactions are catalysed by multiple liver P450 enzymes in vitro in isolated rat liver microsomes. The present pharmacokinetic study investigates the potential for modulation of these alternative pathways of IF metabolism in vivo using the adult male Fischer 344 rat model. Rats were treated with IF alone or in conjunction with various P450 inducers and inhibitors in an effort to improve the balance between drug activation and drug inactivation. Plasma concentrations, areas under the curve (AUC) and half-lives were calculated for 4-OH-IF and CA, allowing estimations of the extent of IF activation and deactivation/toxification. Induction of liver P450 2B enzymes by 4-day high-dose phenobarbital (PB) pretreatment significantly decreased the fraction of IF undergoing 4-hydroxylation (AUC(4-OH-IF)/AUC(4-OH-IF)+AUC(CA)), from 37% to 22% of total metabolism (P < 0.05), consistent with in vitro findings that the PB-inducible P450 enzyme 2B1 plays a major role in IF N-dechloroethylation. Pretreatment with the P450 3A inducer dexamethasone proportionally decreased the AUC for both IF metabolites, without any net impact on the fraction of IF undergoing metabolic activation. By contrast, the P450 2B1 inhibitor metyrapone preferentially increased the AUC for the 4-hydroxylation pathway in 3-day low-dose PB-induced rats, thereby increasing the total fraction of IF metabolized via the activation pathway from 36% to 54% (P < 0.05), whereas the P450 inhibitors orphenadrine and troleandomycin had no significant affect on AUC values. These findings demonstrate specific roles for P450 2B and 3A enzymes in catalysing these pathways of IF metabolism in vivo, and demonstrate the potential for modulation of IF's alternative metabolic pathways in a therapeutically useful manner. These studies also highlight several clinically relevant drug interactions that may occur during concomitant administration of IF with drugs and other compounds that modulate hepatic P450 enzyme levels.

Animals↗
PubMed1997

A nonviral vector system for efficient gene transfer to corneal endothelial cells via membrane integrins.

BACKGROUND: Genetic manipulation of allografts to suppress their ability to induce rejection is a promising approach for controlling rejection responses. A key to this approach is the development of appropriate DNA vectors. We are developing nonviral DNA vector systems based on synthetic peptides containing an integrin-binding segment for cellular targeting and a polylysine segment for DNA binding. METHODS: Two such peptides have been tested for their ability to deliver the beta-galactosidase reporter gene to the corneal endothelial cells of the rabbit, pig, and man. One peptide was derived from a phage display library, the other from the integrin-binding moiety of the toxin from the American pit viper, Crotalus molossus molossus. Corneas were cultured overnight and then exposed to the DNA/peptide vector under a variety of conditions involving different DNA concentrations, chloroquine concentrations, times of exposure, presence of serum, and presence of polyanion buffers. Expression of the beta-galactosidase gene was determined after 3 additional days in culture. Effects of the treatment on the viability of the endothelium were examined by confocal microscopy. RESULTS: We report that approximately 30% of corneal endothelial cells can be transfected with our optimal protocol using the molossin-based vector. Transfection is dependent on the presence of chloroquine and is inhibited by polyanion buffers such as HEPES. Viability of the corneal endothelium was excellent, except if corneas were incubated at high concentrations of chloroquine (0.5 mM) for prolonged periods (24 hr). CONCLUSIONS: Synthetic peptides containing both an integrin targeting and a DNA-binding moiety are promising as simple and highly versatile DNA vectors for use in corneal transplantation.

Animals↗
PubMed1997

Discordant expression of major histocompatibility complex class II antigens and invariant chain in interstitial dendritic cells. Implications for self-tolerance and immunity.

BACKGROUND: The invariant chain plays a crucial role in antigen presentation by influencing the expression and peptide loading of major histocompatibility complex (MHC) class II molecules. Therefore, coordinate expression of these molecules is important for antigen presentation. METHODS: Immunohistological studies were performed on frozen sections of many rat tissues in order to examine expression of invariant chain and MHC class II antigens. RESULTS: Although coordinately regulated in most tissues, the interstitial dendritic cell (and the renal tubular epithelial cell) was always negative for invariant chain, while strongly positive for MHC class II antigens. However, renal tubular epithelial cells strongly expressed invariant chain during kidney graft rejection. CONCLUSIONS: The absence of invariant chain in interstitial dendritic cells is unexpected, in view of their presumed function as sentinel antigen-presenting cells in the connective tissues. This might have important implications for antigen presentation for tolerance and immunity.

Animals↗
PubMed1997

Toxoplasma gondii infection in the mountain hare (Lepus timidus) and domestic rabbit (Oryctolagus cuniculus). I. Pathology.

Mountain hares and domestic rabbits infected experimentally with Toxoplasma gondii were compared in respect of pathological changes and distribution of organisms in the tissues. Seven hares and nine rabbits were each inoculated orally with 50 oocysts of the T. gondii Tg-SweF1 isolate and killed after one week to avoid adverse clinical effects. The only clinical sign observed was respiratory distress in a single hare. At necropsy, gross lesions, which occurred only in the hares, were found in the mesenteric lymph nodes and occasionally the liver. Histologically, the hares showed extensive necrotic areas in the small intestine, mesenteric lymph nodes and liver, and less prominent foci of necrosis in various other organs. Immunohistochemically, T. gondii was identified in all organs examined, large numbers of organisms being associated with the lesions. These findings were analogous to those seen previously in naturally infected hares. In the rabbit, the pathological changes consisted mainly of accumulations of mononuclear cells in the liver and heart, and T. gondii was found only in the heart and skeletal muscle of two rabbits. Thus, the hares developed severe acute toxoplasmosis, but the rabbits showed few organisms and no major tissue damage one week after inoculation.

Animals↗
PubMed1997

Toxoplasma gondii infection in the mountain hare (Lepus timidus) and domestic rabbit (Oryctolagus cuniculus). II. Early immune reactions.

As already reported, the mountain hare is much more susceptible than the domestic rabbit to oral inoculation with Toxoplasma gondii, as judged by pathological changes and dissemination of parasites within the body. In the present paper, further interspecies variations are reported. Concentrations of the acute phase reactant haptoglobin were raised in hares but not in rabbits one week post-infection (pi), probably reflecting the severe tissue damage present. No difference in the early humoral immune response of hares and rabbits was found, both species producing IgM and IgG antibodies to T. gondii one week pi. Lymphocyte stimulation tests performed before and one week after inoculation showed a high proliferative response to the parasite in blood cell cultures from rabbits but not hares. The fatal outcome of T. gondii infection in the hares is probably due, at least in part, to the lack of cellular response.

Animals↗
PubMed1997

Organ culture of arteries for experimental studies of vascular endothelium in situ.

The objective of this study was to determine whether organ culture of arteries could be used as a more physiological model than endothelial cell culture for the study of vascular endothelium in vitro. Small pieces of artery from rat, pig, piglet and man were cultured in 24-well plates for up to seven or eight days to study the characteristics of the vascular endothelial cell layer during the first week of culture, in particular its integrity, viability and propensity for cell division. Using conventional and confocal microscopy, silver-stained endothelial cell boundaries were shown to be intact at all time points, up to and including day 7. However, occasional very small gaps between endothelial cells were seen with the scanning electron microscope under high power at day 7. Using the bromodeoxyuridine technique, no endothelial cell division was seen at day 4 in any species, except for the occasional endothelial cell in rat aorta. At day 7, pig, piglet and human arteries showed only very occasional dividing endothelial cells, but many endothelial cells had divided by day 7 in rat aorta. Viability of the endothelium was assessed using fluorochromes and examination of the endothelial layer en face using confocal microscopy. Viability was always excellent (> 95%) up to day 4. By day 7, occasional patches of dead cells could be seen, which were most obvious in rat aorta. This study demonstrates that endothelial cells can be studied in situ in organ culture with intact morphology, lack of cell division and excellent viability for a minimum of four days. For many research questions involving vascular endothelium--for example the pathophysiology of hyperacute rejection--short-term organ culture of vessels is likely to represent a more physiological model than endothelial cell culture.

Animals↗
PubMed1997

Integrin-mediated transfection with peptides containing arginine-glycine-aspartic acid domains.

Two synthetic peptides comprising an RGD moiety for integrin binding and a polylysine moiety for DNA binding were tested for transfection efficiency under a variety of different conditions. Binding of target cells to the peptide was shown to be strongly dependent on cyclisation of the peptides via cysteine residues. Low (10 microM) concentrations of chloroquine, added to assist endocytic exit, unexpectedly reduced transfection efficiency in two of the cell lines tested. COS-7 and ECV304. However, transfection efficiency increased at higher chloroquine concentrations and exceeded that in the absence of chloroquine in the case of the COS-7 and A375M cell lines. With the ECV304 cell line, optimum transfection occurred in the absence of chloroquine. Transfection efficiency of the peptides was greatest at peptide:DNA ratios of 4:1 (w/w), which were calculated to generate complexes containing approximately 5000 peptide molecules per plasmid. This represented approximately a 6:1 ratio of positive to negative charges. Peptide 5 was shown to have a higher transfection efficiency under most conditions, possibly because of more efficient stabilisation of cyclisation by two cysteine-cysteine bonds.

Animals↗