Biomedical subjects

J N Kearney

Publications and source records attributed to J N Kearney.

At least 37 records · Page 2Linked to original sources

PubMed1996

Assessment of immunogenicity and viability of homologous human cardiac valves in vitro.

Degenerative changes in a proportion of implanted homologous cardiac valves are considered to be due to the immunologic tissue reactions initiated by the donor valves. Sterilization and storage protocols can be used to modulate the immunogenicity of donor valves prior to implantation. Therefore, it is essential to assess the effect of treatment protocols on the immunogenicity and viability of donor valve tissue. The optimal conditions for two novel in vitro tests to assess the immunogenicity and viability of human cardiac valve tissue, the valve cusp cell/responder lymphocyte reaction to assess immunogenicity and the tetrazolium based colorimetric assay to assess viability of valve tissue, are described. The in vitro tests that have been developed in this study will be helpful in assessing the effect of various treatment protocols on the immunogenicity and viability of homologous human cardiac valves.

Analysis of Variance↗

PubMed1995

Cytokine and protease levels in healing and non-healing chronic venous leg ulcers.

Leg ulcers present a common and recurring problem in older people creating discomfort and distress for the patient and a great cost to the health care services. Cultured keratinocyte grafts have been used by many investigators to stimulate healing of chronic venous ulcers. It has been proposed that they may do this by producing cytokines which modulate the healing process. However, the types and levels of cytokines in the leg ulcer fluid before and during healing are not known. Wound fluid was collected from venous leg ulcers in 18 patients beneath occlusive Tegaderm dressing for 4 to 6 h. The leg ulcers were divided on clinical criteria into 'healing' and 'non-healing'. PDGF-AB, GM-CSF, IL-1 alpha, IL-1 beta, IL-6 and bFGF were measured by ELISA and the levels of IL-1 alpha, IL-1 beta and IL-6 were also measured using biological assays. The effect of leg ulcer wound fluid on fibroblast and keratinocyte proliferation was measured indirectly by 3H-thymidine incorporation and MTT assay. Total protein, albumin levels, fibronectin degrading activity and collagenase activity, both active and latent were measured. No statistically significant differences in the levels of cytokines or collagenase were identified between healing and non-healing leg ulcers in the sample of leg ulcers studied. However, this study does give valuable information concerning the levels of cytokines and collagenase in chronic leg ulcer wound fluid.

Aged↗

PubMed1995

Calcium: a crucial consideration in serum-free keratinocyte culture.

This investigation was conducted when previously repeatable experimental data became impossible to reproduce when using keratinocytes cultured in serum-free medium. Differences in calcium molarity between batches of medium were identified as a source of variation in cultured keratinocyte populations. The susceptibility of cultured keratinocytes to even small alterations in calcium molarity has been demonstrated. 2 regular medium batches were compared with a special preparation of medium, devoid of calcium chloride then supplemented with a known concentration of calcium ions. Culture progress in each medium was assessed by: morphological observation, % cells expressing involucrin and proliferating cell nuclear antigen, cell attachment, growth rate and colony forming efficiency. In order to control the phenotype of cultured keratinocytes, in a reproducible system, it is recommended that serum-free keratinocytes medium is purchased with the omission of calcium chloride. Supplementation of this medium may then be made by the investigator to suit individual culture requirements.

Calcium↗

PubMed1995

In vitro modulation of keratinocyte-derived interleukin-1 alpha (IL-1 alpha) and peripheral blood mononuclear cell-derived IL-1 beta release in response to cutaneous commensal microorganisms.

The ability of a range of skin commensal microorganisms to modulate interleukin-1 (IL-1) release by cultured human keratinocytes and peripheral blood mononuclear cells (PBMCs) was investigated by a combination of enzyme-linked immunosorbent assays and bioassays. Three fractions (formaldehyde-treated whole cells, culture supernatants, and cellular fractions) were prepared from Propionibacterium acnes, Propionibacterium granulosum, Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus hominis, and Malassezia furfur serovar B. The levels of immunochemical IL-1 alpha released by cultured keratinocytes during coincubations with these microbial fractions ranged from 0 to 136 pg/ml and were maximal after 72 h. No microbial fraction consistently upregulated immunochemical IL-1 alpha release by freshly isolated keratinocytes from two donors and a transformed cell line, all of which produced the cytokine constitutively to various extents. Bioassays revealed that most of the IL-1 released was biologically inactive. In contrast, whole cells of formaldehyde-treated P. granulosum and S. epidermidis significantly stimulated release of IL-1 beta by PBMCs from three donors compared with the negative control (culture medium). Release was maximal at 24 h. Coincubation with intact cells of the yeast M. furfur significantly decreased levels of IL-1 beta below the values for the negative control by PBMCs from all three donors. There was good correlation between bioassay data and immunoassay data for IL-1 beta, and the depressive effect of M. furfur cells on cytokine production by all three cultures of PBMCs was mirrored in the levels of bioactive cytokine. This reduction in IL-1 beta release by PBMCs by M. furfur may provide an explanation why dermatoses thought to be caused by this yeast are essentially noninflammatory or only mildly inflammatory.

Humans↗

PubMed1994

In vitro immunogenicity of allogeneic cardiac valves.

The immunogenicity of allogeneic cardiac valves (ACV) has not been previously demonstrated in vitro, though valve failure due to tissue degeneration has been attributed to adverse immunological reactions. A novel in vitro assay has been developed in a Brown Norway (BN; RT1n)-Lewis (RT1; donor-recipient) rat model system that demonstrates the immunogenicity of ACVs. A single cell suspension of viable cardiac valve conduit (CVC) cells was obtained by collagenase treatment of BN rat aortic valve conduits. Brown Norway rat CVC cells (5 x 10(4)) and Lewis responder lymphocytes (10(5)) were co-cultured in 96 well plates in RPMI 1640 plus 2.5% (v/v) non heat-inactivated Lewis rat serum and supplements with appropriate controls. Stimulation of responder lymphocytes by CVC cells was measured by 3H-thymidine incorporation into DNA. The counts obtained between 96-192 h of co-culture in the CVC cell/responder lymphocyte reaction were significantly higher (P < 0.05) than those of responder cell controls as assessed by analysis of variance. These results indicate the presence of potent immunostimulatory cells in viable ACVs and the possibility of using a sensitive and reproducible in vitro assay to evaluate ACV immunogenicity.

Animals↗

PubMed1993

The effects of variation of cryopreservation protocols on the immunogenicity of allogeneic skin grafts.

The use of allografts to effect wound closure on full thickness skin injuries is limited by unpredictable graft rejection times. If the period of graft take could be extended, the use of allografts would reduce the morbidity and mortality associated with these wounds. This study evaluated the effects of variation in the cryopreservation protocol on the viability and immunogenicity of skin using a murine model system. Immunogenicity was assessed by the stimulatory activity of C3H (H-2K) skin-derived epidermal cells (EC) in primary one-way EC/lymphocyte reactions with BALB/c (H-2d) and CBA (H-2K) responder lymphocytes. Viability was determined by measuring tetrazolium reductase activity. The following cryopreservation protocols were assessed: freezing at 1, 30, 64, and > 100 degrees C/min in 10 and 15% (v/v) Me2SO and freezing at 30 degrees C/min in 5 to 20% (v/v) Me2SO or glycerol. A cryopreservation protocol of 30 degrees C/min in 15% (v/v) Me2SO proved optimal for murine skin allograft storage and immunomodulation. The viability of skin treated by this protocol was maintained (78% of fresh skin viability, no significant difference analysis of variance). The stimulatory capacity of treated EC for H-2K and H-2d lymphocytes was 5 +/- 4 and 5 +/- 9% (+/- 95% confidence limits) of fresh EC (100%) activity. Langerhans cell numbers in epidermal sheets and EC suspensions did not correlate with the stimulatory capacity of fresh and treated EC for allogeneic lymphocytes. A functional impairment of Langerhans cell immunostimulatory capacity was implied.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Ethylene oxide sterilisation of allogenic bone implants.

We have evaluated the ability of ethylene oxide gas to penetrate bone matrices and subsequently kill a defined microbiological load. These experiments demonstrated that freeze-dried cancellous and cortical bone did not pose a barrier to the rapid diffusion of the gas. A normal cycle on a commercial ethylene oxide steriliser provided a sixfold overkill. Minor differences in the rate at which the microbiological load was killed (D-value) were seen between cortical and cancellous bone, processed and unprocessed bone, and bone of different thickness. These differences were negligible in comparison to the degree of overkill observed. Spore indicators placed freely in the steriliser chamber were shown to act as excellent monitors for the bone sterilisation process. Preliminary studies of ethylene oxide residuals indicated that processed, freeze-dried bone could be sterilised without leaving leachable toxic residuals in the matrix.

Analysis of Variance↗

PubMed1993

Evaluation of collagen cross-linking techniques for the stabilization of tissue matrices.

The use of glutaraldehyde (GTA) for cross-linking biological tissue implants has a number of undesirable side effects, including cytotoxicity and induction of calcification. In an attempt to find an improved cross-linking agent for tissue implants, we have evaluated a number of cross-linking procedures shown previously to be effective with pure collagen or collagenous substrates, including GTA, succinic anhydride, cyanamide, 1-ethyl-3-(dimethylaminopropyl)carbodi-imide, dimethyl suberimidate, ascorbate-copper, glucose-lysine and acyl azide treatments. Apart from GTA, only acyl azide treatment significantly cross-linked human dermis, the degree of cross-linking being better than that seen after treatment of dermis with 1 mM GTA. Acyl azide treatment of thicker vascular tissue (porcine aorta) also resulted in a significantly cross-linked tissue. Preliminary cytotoxicity studies suggested that acyl azide treatment was not toxic, and, therefore, coupled with its cross-linking ability, this treatment is worthy of further investigation and may prove to be an alternative to GTA for the treatment of bioprostheses.

3T3 Cells↗

PubMed1992

Bone banks.

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Bone and Bones↗

PubMed1992

The hydrodynamic function and leaflet dynamics of aortic and pulmonary roots and valves: an in vitro study.

Fresh homograft aortic and pulmonary roots were tested in a pulsatile flow simulator to assess their hydrodynamic function and leaflet opening characteristics. Simultaneous flow and pressure measurements were obtained for a range of cardiac outputs. The effective orifice area and regurgitant volumes were calculated. The mean pressure difference across the pulmonary roots was obtained under both left and right side pressures. A video recording of valve leaflet function was also obtained for each valve. A comparison was made with four porcine bioprosthetic heart valves (21 and 23 mm). The mean pressure difference with respect to flow for the 21 mm Hancock II and Intact porcine bioprostheses was significantly higher than that for all aortic roots tested. The 24 mm aortic root showed significantly lower pressure drop compared to all porcine valves tested. The mean pressure difference across each pulmonary root at pulmonary pressures was significantly greater than at systemic pressures. At systemic pressures the fully open leaflets had a triangular orifice with low leaflet open bending strains at the commissures. At the lower internal pressures, with reduced dilation of the root, higher bending strains were noted. These were not as severe as seen in porcine valves.

Adult↗

PubMed1991

Evaluation of NaOH treatment of human dura mater implants to obviate Creutzfeldt-Jakob disease transmission.

One of the few disinfectants known to inactivate the causative agent of Creutzfeldt-Jakob Disease is NaOH. In this study, NaOH was evaluated as a possible routine treatment for human dura mater alloimplants. Use of high concentration NaOH (1 M) resulted in protein loss and macroscopic changes to the tissue. Lower concentrations (0.1 M), although exhibiting little direct detrimental effect, greatly increased the susceptibility of the tissue to collagenase digestion. The use of NaOH treated commercial or institutionally prepared human dura mater should be approached with caution.

Creutzfeldt-Jakob Syndrome↗

PubMed1991

Cryopreservation of cultured skin cells.

Cryopreservation of human skin keratinocytes and fibroblast cells has been evaluated using three cryoprotectants at various concentrations, three cooling rates and two warming rates. Optimal combinations of factors were determined. Selection for tolerance to cryopreservation was not detected after repeated freeze/thaw cycles. A beneficial effect of serum was shown to be cooling rate dependent.

Burns↗

PubMed1991

The effects of minocycline and tetracycline on the mitotic response of human peripheral blood-lymphocytes.

The effects of minocycline and tetracycline on the mitotic response of human peripheral blood lymphocytes was investigated in vitro. The effects of the antibiotics on the mitotic response of purified lymphocytes stimulated with Interleukin-1 beta varied according to the individual from whom the lymphocytes were obtained. At concentrations above those reported to be present in serum during conventional therapy (2-8 mg/l), there was a tendency for both minocycline and tetracycline to suppress the mitotic response. Minocycline was superior to tetracycline in this respect. However, at physiological concentrations the antibiotics either had no significant effect, suppressed the mitotic response (minocycline at 2 mg/l with one of six donors), or enhanced the mitotic response (tetracycline at 2 and 8 mg/l with four of six donors). The stimulatory effect of tetracycline was not demonstrated when lymphocytes were cultured in whole blood for up to seven days with the antibiotic alone. Similar effects of the antibiotics were observed when mononuclear cell fractions isolated from six donors were stimulated with an optimal concentration of phytohaemagglutinin (PHA). Stimulation of lymphocytes in whole blood cultures with PHA in the presence of minocycline and tetracycline revealed that, under these culture conditions, the antibiotics could suppress the mitotic response of lymphocytes at physiological doses with cells from a majority of donors.

Adult↗

PubMed1991

Hydrodynamic function of second generation porcine bioprosthetic heart valves.

The hydrodynamic function and leaflet dynamics of second generation porcine valves prepared with low- or zero-pressure fixation have been studied and compared to first generation porcine bioprostheses, bileaflet, and tilting disc mechanical valves. The Carpentier-Edwards Supra-Annular and Hancock II valves showed lower pressure drops than the Medtronic Intact valve and first generation porcine valves, and comparable overall energy losses to mechanical valves at normal cardiac outputs. Only the zero-pressure fixed Intact valve showed synchronous leaflet opening. Delayed leaflet opening and high opening pressures were found in both low- and high-pressure fixed porcine valves. All porcine bioprostheses showed high open leaflet bending strains. Fixation of valve leaflets with "near zero" pressure fixation and a more physiological neutral geometry is necessary to ensure synchronous leaflet opening at low flows and a reduction in commissural bending strains.

Biomechanical Phenomena↗

PubMed1990

Effects of cryobiological variables on the survival of skin using a defined murine model.

Skin from an inbred strain of hairless mouse was used as a homogeneous model tissue for studies of skin cryopreservation. Tetrazolium reductase enzyme activity was used to assess tissue viability. Hepes-buffered 199 tissue culture medium was confirmed to be a suitable basal medium, to which cryoprotectants were added. Addition of serum to the cryoprotective cocktail had no beneficial effect. Three cryoprotectants, dimethyl sulfoxide, ethanediol, and glycerol were evaluated. There was no evidence of specific toxicity attributable to the cryoprotective agents during the permeation period; however, short permeation times at low temperature were associated with maximum skin viability. Following freezing and thawing, higher viabilities were obtained when using a slow (-1 degree C min-1) or medium (-60 degree C min-1) rather than a fast (immersion in liquid nitrogen) cooling rate. Dimethyl sulfoxide was a marginally better cryoprotectant overall, although this difference was not statistically significant.

Animals↗

PubMed1990

Cryopreservation of skin using a murine model: validation of a prognostic viability assay.

In order to evaluate the many variables that can affect cryopreservation success, a simple, highly reproducible model system is required. We have evaluated the use of tetrazolium reductase activity as a prognostic indicator of skin viability in an inbred murine model. Two inbred hairless mouse strains were characterized in studies on autografting and allografting following different skin-storage protocols. Skin tetrazolium reductase (TR) activity correlated well with oxygen consumption, and with graft success--the ultimate performance criterion--following varying degrees of cryogenic injury. The assay was shown to be highly reproducible. In a series of factorial experiments the only factors affecting TR activity were those concerning the mouse donors, i.e., mouse strain, age, sex, and body area. The effects of these factors on TR activity were fully characterized.

Animals↗

PubMed1990

Fabrication and reorganization of dermal equivalents suitable for skin grafting after major cutaneous injury.

The incorporation of fibroblasts into a hydrated collagen lattice results in lattice contraction and collagen reorganization to form a dermal equivalent. Lattices fabricated with 7.7 mg collagen and seeded with 1 X 10(5) cells were found to give the best results in terms of their mechanical properties and ability to maintain cell viability. Newly-cast lattices were found to be completely digested by 0.085 units/ml bacterial collagenase in 3 h, whereas after 30 d in culture, limited digestion took place over 24 h. Electrophoretic analysis showed that the proportion of cross-linked collagen in the 30 d lattice was increased by 2.5-fold compared to the initial collagen preparation. These results indicate that a dermal equivalent better suited for grafting may be produced after 20-30 d in culture.

Animals↗

PubMed1990

The effect of bacterial products on human fibroblast and keratinocyte detachment and viability.

An in vitro model has been developed to study the effect of soluble bacterial products on the viability and detachment of skin cell types utilized cultured grafts. Microbial products prepared from clinical isolates of bacterial species which most commonly colonize burn lesions showed marked variation in their ability to detach and kill both keratinocytes and fibroblasts. All three isolates of Acinetobacter spp. tested were effective in causing detachment and death of keratinocytes and fibroblasts, whereas Escherichia coli, Proteus mirabilis and Enterobacter spp. tested had little, or no, effect on detachment or viability for either skin cell type. Four Staphylococcus aureus isolates elicited variable strain-dependent results with regard to detachment and viability. One isolate possessed activity specific for keratinocyte detachment and death. These results indicate the possible undesirable effects such bacterial species may have on graft success in colonized burn wounds.

Bacteria↗