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Biomedical subjects

E Ingham

Publications and source records attributed to E Ingham.

At least 109 records · Page 6Linked to original sources
PubMed1996

Ultra-high molecular weight polyethylene wear debris generated in vivo and in laboratory tests; the influence of counterface roughness.

The objective of this study was to investigate the effect of counterface roughness and lubricant on the morphology of ultra-high molecular weight polyethylene (UHMWPE) wear debris generated in laboratory wear tests, and to compare this with debris isolated from explanted tissue. Laboratory tests used UHMWPE pins sliding against stainless steel counterfaces. Both water and serum lubricants were used in conjunction with rough and smooth counterfaces. The lubricants and tissue from revision hip surgery were processed to digest the proteins and permit filtration. This involved denaturing the proteins with potassium hydroxide (KOH), sedimentation of any remaining proteins, and further digestion of these proteins with chromic acid. All fractions were then passed through a 0.2 micron membrane, and the debris examined using scanning electron microscopy. The laboratory studies showed that the major variable influencing debris morphology was counterface roughness. The rougher counterfaces produced larger numbers of smaller particles, with a size range extending below 1 micron. For smooth counterfaces there were fewer of these small particles, and evidence of larger platelets, greater than 10 microns in diameter. Analysis of the debris from explanted tissues showed a wide variation in the particle size distribution, ranging from below 1 micron up to several millimetres in size. Of major clinical significance in relation to osteolysis and loosening is roughening of the femoral components, which may lead to greater numbers of the sub-micron-sized particles.

Adult↗
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↗
PubMed1994

The effect of cytokines on the in-vitro erythropoietin bioassay.

The effects of various concentrations of granulocyte macrophage colony stimulating factor (GM-CSF), gamma interferon (gamma IFN) and interleukins 1 alpha (IL-1 alpha), 1 beta (IL-1 beta) and 3 (IL-3) on the anaemic mouse spleen cell bioassay for erythropoietin (EPO) were investigated. Addition of IL-3 and GM-CSF at various concentrations had no effect on EPO stimulated 3H thymidine incorporation. However the addition of IL-1 alpha, IL1-beta and gamma IFN (3.3 x 10(-8) gl-1) caused a significant (P < 0.01) inhibition of EPO stimulated thymidine incorporation. This suggests that the EPO bioassay may be influenced by variable levels of some inflammatory cytokines in serum. Previous studies have shown that the bioassay is influenced by serum transferrin levels and thus serum immunoassays remain the technique of choice for specific estimates of EPO. Since EPO bioassays are not specific, they should be reserved for situations in which an estimate of the total erythropoietic activity of serum is required.

Animals↗
PubMed1994

Cell-mediated immune responses to Malassezia furfur serovars A, B and C in patients with pityriasis versicolor, seborrheic dermatitis and controls.

It has been postulated that patients with Malassezia furfur-associated dermatoses have a deficient cell-mediated immune response to M. furfur. This study examined the cell-mediated immune responses to M. furfur serovars A, B and C of 10 patients with pityriasis versicolor and 10 age- and sex-matched controls; and 10 patients with seborrheic dermatitis and 10 age- and sex-matched controls. The responses to each serovar of M. furfur were assessed using the lymphocyte transformation assay and the leukocyte migration inhibition assay. The lymphocyte transformation responses of the patients with pityriasis versicolor to M. furfur serovars A, B and C (0/10, 6/10 and 5/10 respectively) were not significantly different from those of controls (0/10, 2/10 and 1/10). However, for patients with seborrheic dermatitis, significantly more patients' lymphocytes responded to serovars B and C (6/10 and 6/10 respectively) than those of controls (1/10 and 1/10). No patient or control responded to serovar A. In the leukocyte migration inhibition assay, the leukocytes from a greater proportion of patients with pityriasis versicolor (5/7) responded to serovar B than controls (2/10); and the leukocytes from a greater proportion of patients with seborrheic dermatitis (4/10) responded to serovar C than controls (0/9). Thus, this data did not indicate the presence of any cell-mediated immune deficiency to M. furfur in patients with pityriasis versicolor or seborrheic dermatitis, as measured by the lymphocyte transformation assay or the leukocyte migration inhibition assay. The greater responsiveness of T lymphocytes from patients may indicate that T lymphocytes might be involved in the pathogenesis of these diseases.

Adult↗
PubMed1994

Humoral immunity to Malassezia furfur serovars A, B and C in patients with pityriasis versicolor, seborrheic dermatitis and controls.

This study examined the humoral immune responses to Malassezia furfur serovars A, B and C of 10 patients with pityriasis versicolor, 10 patients with seborrheic dermatitis and 20 age- and sex-matched controls. A transferable solid-phase ELISA was used to determine titres of total Igs, IgM, IgA and IgG specific to M. furfur serovars A, B and C. The results demonstrated that patients with seborrheic dermatitis had a significantly higher titre of total Igs to serovar A than patients with pityriasis versicolor; and that patients with seborrheic dermatitis had a significantly higher titre of IgA to serovar C than patients with pityriasis versicolor. The titres of total Igs for controls and patients with seborrheic dermatitis were significantly lower to serovar B than to serovar C. A modified TSP ELISA was used to determine the titres of the IgG subclasses. Titres of IgG1,3,4 to serovar B were significantly higher in seborrheic dermatitis patients than pityriasis versicolor patients and titres of IgG3 to serovar A were significantly higher in seborrheic dermatitis patients than pityriasis versicolor patients. However, despite the differences between the patient groups, none of these results was significantly different to those of controls. Thus, this study did not demonstrate any differences in humoral immunity of patients suffering from Malassezia-associated dermatoses when compared to normal controls. These results may suggest that the humoral immune response to M. furfur is not related to the pathogenesis of Malassezia-associated dermatoses, but simply to the carriage of M. furfur on the skin.

Adult↗
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

Modulation of comedonal levels of interleukin-1 in acne patients treated with tetracyclines.

To understand the basis for the anti-inflammatory activity of tetracyclines in acne, we compared the cytokine profiles [interleukin 1 (IL-1) alpha and beta, tumor necrosis factor (TNF) alpha, and IL-6] and bacterial flora of 66 open comedones removed from eleven patients before and after at least 8 weeks treatment with either tetracycline or minocycline. Pre-treatment, the only cytokine regularly recovered from comedones was bioactive IL-1 alpha-like material. The mean concentration of IL-1 alpha-like bioactivity/mg comedonal material rose from 272.0 +/- 88.6 pg to 844.3 +/- 196.7 pg following treatment (p < 0.05, Wilcoxon matched pairs). All six minocycline-treated patients showed an increase in bioactive IL-1 alpha-like material compared with three of five tetracycline-treated patients. The incidence (p < 0.001, chi 2) and concentration (p < 0.05, Wilcoxon) of immunochemical IL-beta were also raised post-treatment, although significantly more patients assigned to minocycline therapy had detectable levels of this cytokine before therapy was initiated. However, the mean concentration of IL-1 beta/mg comedonal material post-treatment was similar in both groups (72.5 +/- 23.3 pg for tetracycline-treated compared with 78.6 +/- 41.9 pg for minocycline-treated patients). The other cytokines were either absent (IL-6) or present in < 10% of comedones (TNF alpha) before and after therapy. Following treatment, only three of 11 patients showed a decrease of > or = 1 log10 in propionibacterial numbers/mg comedonal material, whereas six patients showed an increase of > 0.5 log10 in numbers of staphylococci. In eight patients, the increase or decrease in staphylococcal numbers correlated with the change in concentration of IL-1 alpha-like bioactivity. This is the first study to show an effect of antibiotic therapy on cytokine levels in vivo. Increased levels of IL-1 in comedones destined to become inflamed may enhance resolution and promote repair of the damaged follicular epithelium. Hence, these results provide further evidence of the augmentation of immune responses by tetracyclines and support the hypothesis that epidermal IL-1 plays a physiologic role in wound healing.

Acne Vulgaris↗
PubMed1993

The carriage of Malassezia furfur serovars A, B and C in patients with pityriasis versicolor, seborrhoeic dermatitis and controls.

The aetiological role of Malassezia furfur in various dermatoses is controversial. The role of the three serovars of M. furfur in Malassezia-associated diseases has not been investigated. This study measured population densities of M. furfur serovars A, B and C, propionibacteria and Micrococcaceae on the chest, back, forehead, left and right cheeks of 10 patients with pityriasis versicolor, and 10 age- and sex-matched controls; and 10 patients with seborrhoeic dermatitis, and 10 age- and sex-matched controls. The population densities of M. furfur, propionibacteria and Micrococcaceae did not vary at a given site between patients and the corresponding control subjects. Malassezia furfur serovar A was found to be the predominant isolate on the chest and back of all four groups, but there was no difference in the distribution of serovars on the forehead and cheeks. No serovar was specifically associated with lesional skin in either disease. Thus, this data indicated that there was no difference in either the total population density of M. furfur or the distribution of serovars on lesional skin compared with control skin in either pityriasis versicolor or seborrhoeic dermatitis.

Adult↗
PubMed1992

Pro-inflammatory levels of interleukin-1 alpha-like bioactivity are present in the majority of open comedones in acne vulgaris.

The factors that initiate the inflammatory response in acne are not known. The presence of pro-inflammatory cytokines in acne comedones was therefore investigated. One hundred eight open comedones were collected from 18 untreated acne patients (10 male, 8 female). Each comedone was homogenized and centrifuged, and the supernatant was analyzed for bioactive and immunochemically detectable IL-1 alpha, IL-1 beta, and TNF alpha. Viable counts of propionibacteria, staphylococci, and Malassezia spp. were determined in the comedone pellet. Bioactive IL-1 alpha-like material was demonstrated in 76% of open comedones (range of 23-4765 pg IL-1 alpha-like bioactivity/mg of comedone material). In 58% of comedones, levels exceeded 100 pg/mg. There was no correlation between IL-1 alpha-like bioactivity and IL-1 alpha determined immunochemically. Bioactive IL-1 beta was not detected in any comedones. Twenty-four percent contained low levels of immunochemical IL-1 beta (range 12-103 pg IL-1 beta/mg comedone material). Bioactive TNF alpha was detected in three comedones with a further five comedones containing immunochemical TNF alpha (range of 61-820 pg TNF alpha/mg comedone material). The majority of open comedones (97%) contained microorganisms. There was, however, no significant correlation (Spearman's rank) between levels of any cytokine, in particular IL-1 alpha-like bioactivity, and numbers of microorganisms. Thus, bioactive IL-1 alpha-like material in the majority of open comedones may be concerned in the initiation of inflammation in acne following spongiosis or rupture of the pilosebaceous follicle wall.

Acne Vulgaris↗
PubMed1992

Humoral responses to Malassezia furfur serovars A, B and C in normal individuals of various ages.

A transferable solid-phase (TSP) ELISA was developed for the determination of antibody titres specific to Malassezia furfur serovars A, B and C in human sera. A survey of levels of class-specific antibodies (IgM, IgG and IgA) to M. furfur serovars A, B and C in relation to age (2-64 years; 60 individuals) demonstrated that individuals had immunity to M. furfur by the age of 2-3 years. There was no difference in either IgM or IgG levels into adulthood. The only age-related differences were lower IgM titres to the three serovars in the 60-64 year age-group compared with younger individuals. There was, however, a difference between titres of antibody specific to the three serovars. The mean reciprocal log2 IgM titre to serovar A (6.9) was significantly higher (P < 0.05) than that to serovar B (mean reciprocal log2 titre of 5.8), but not to serovar C (6.1). In contrast, the mean reciprocal log2 IgG titre to serovar A (6.5) was significantly lower (P < 0.05) than those to serovars B and C (mean reciprocal log2 titre of 8.9 in both cases).

Adult↗
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↗
PubMed1990

Modulation of the proliferative response of murine thymocytes stimulated by IL-1, and enhancement of IL-1 beta secretion from mononuclear phagocytes by tetracyclines.

The capacity of minocycline and tetracycline for modulation of IL-1 secretion by LPS-stimulated human monocytes was investigated in vitro. Both minocycline and tetracycline suppressed the murine thymocyte co-mitogenic bioassay of IL-1 at 2 and 4 mg/l respectively. IL-1 beta secretion by LPS-stimulated human monocytes cultured for 24h at 1 x 10(6)/ml with 0, 5, 10 and 50 mg/l minocycline or tetracycline was therefore determined by ELISA. Monocytes from five different individuals served as replicates. LPS-stimulated monocytes secreted significantly more IL-1 beta in the presence of minocycline (P less than 0.01, 2-way analysis of variance). There was no difference in the enhanced levels of IL-1 beta secreted with 5 mg/l minocycline compared with 50 mg/l minocycline. Loss of viability could only be associated with enhanced IL-1 beta release by monocytes with 50 mg/l minocycline. Although tetracycline enhanced IL-1 beta secretion in four of the five replicate experiments, this did not prove significant owing to the large error variance between individual monocyte cultures. Thus, therapeutic levels of tetracyclines, especially minocycline, modulate mononuclear cell activities in vitro.

Adult↗
PubMed1990

Differentiation of three serovars of Malassezia furfur.

Malassezia furfur strains were isolated from the clinically normal skin of 10 volunteers by swabbing four different sites (forehead, ear, back and chest). The strains could be divided into three basic groups on the basis of cultural characteristics. Both unabsorbed and absorbed specific rabbit antisera were prepared against nine of the strains, and both species and group specific antigens could be demonstrated. Serologically, three group specific surface antigens could be identified which corresponded to the three groups identifiable on cultural characteristics. The relevance of these findings to previous in vitro results is discussed.

Adult↗