Johann M. Zdolsek

Histamine release and fibrinogen adsorption mediate acute inflammatory responses to biomaterial implants in humans

BackgroundMedical implants often fail as a result of so-called foreign body reactions during which inflammatory cells are recruited to implant surfaces. Despite the clinical importance of this phenomenon, the mechanisms involved in these reactions to biomedical implants in humans are not well understood. The results from animal studies suggest that both fibrinogen adsorption to the implant surface and histamine release by local mast cells are involved in biomaterial-mediated acute inflammatory responses. The purpose of this study was to test this hypothesis in humans.MethodsThirteen male medical student volunteers (Caucasian, 21–30 years of age) were employed for this study. To assess the importance of fibrinogen adsorption, six volunteers were implanted with polyethylene teraphthalate disks pre-coated with their own (fibrinogen-containing) plasma or (fibrinogen-free) serum. To evaluate the importance of histamine, seven volunteers were implanted with uncoated disks with or without prior oral administration of histamine receptor antagonists. The acute inflammatory response was estimated 24 hours later by measuring the activities of implant-associated phagocyte-specific enzymes.ResultsPlasma coated implants accumulated significantly more phagocytes than did serum coated implants and the recruited cells were predominantly macrophage/monocytes. Administration of both H1 and H2 histamine receptor antagonists greatly reduced the recruitment of macrophages/monocytes and neutrophils on implant surfaces.ConclusionIn humans – as in rodents – biomaterial-mediated inflammatory responses involve at least two crucial events: histamine-mediated phagocyte recruitment and phagocyte accumulation on implant surfaces engendered by spontaneously adsorbed host fibrinogen. Based on these results, we conclude that reducing fibrinogen:surface interactions should enhance biocompatibility and that administration of histamine receptor antagonists prior to, and shortly after, medical device implantation should improve the functionality and longevity of medical implants.

Visualization of iron in cultured macrophages: A cytochemical light and electron microscopic study using autometallography ☆

The objective of this study was to develop a sensitive cytochemical method for the visualization of iron, both at light microscopical (LM) and at electron microscopical (EM) levels, in glutaraldehyde-fixed cultured cells with reasonable morphological preservation. The method is based on autometallography (also called the sulfide silver method or the Timm technique). Gold, silver, and various metal sulfides have previously been shown to act as catalysts for cellular silver deposition from a physical developer (autometallography). In our modification of this cytochemistry, a high pH is used during the initial sulfidation step to guarantee adequate levels of sulfide ions to generate enough Fe(II or III) sulfide. Since this procedure may cause severe cellular distortion, we initially stabilize the cultured cells by a glutaraldehyde fixation. We have compared our new high pH, high S2- LM and EM variety of autometallography with other modifications of this technique that have previously been used for LM and EM demonstration of easily sulfidated heavy metals, such as zinc. Cultured mouse macrophages were examined for the localization of reactive metals following endocytosis of ferritin or inorganic Fe(III) iron. Ag-precipitates, presumed to indicate the presence of iron, were predominantly found within secondary lysosomes of the acidic vacuolar apparatus. The relation of the Ag-precipitates to iron was proven by the fact that iron-exposed cells showed a much reduced amount of silver precipitates after subsequent exposure to deferoxamine a potent iron chelator. Moreover, control macrophages neither exposed to iron nor to ferritin showed only a low normal lysosomal content--and a few extralysosomal sites--of reactive substances, believed to be iron.

Identification of Helicobacter pylori DNA in Human Cholesterol Gallstones

Background: The gallbladder mucosa secretes hydrogen ions and is covered by mucus. The environmental conditions for bacterial colonization are similar to those in the stomach. Gallbladder stones often contain DNA from enteric bacteria, but no compelling evidence demonstrates that Helicobacter spp. have been present. The aim of this study was to establish bacterial DNA profiles in cholesterol gallstones with special reference to Helicobacter pylori. Methods: Cholesterol gallstones from 20 patients were subjected to polymerase chain reaction, bacterial profiling by temporal temperature gradient gel electrophoresis, automated DNA sequencing, and Southern blot analysis using a Helicobacter sp. specific primer. A nested ureI- PCR assay was used to discriminate between gastric and non-gastric H. pylori. Results: TTGE, partial 16S rDNA sequencing, and hybridization analysis revealed the presence of DNA presumably representing a mixed bacterial flora in cholesterol gallstones, including H. pylori in the gallstone centres in 11 out of 20 patients. In three cases, the ureI -PCR assay revealed non-gastric H. pylori. Conclusions: These data support the presence of DNA from a mixed bacterial population, including H. pylori in cholesterol gallstones, reflecting either that H. pylori is an indigenous part of a flora in the stone-containing gallbladder or, alternatively, that H. pylori colonization in the biliary tract predisposes to cholesterol gallstone formation.

Acridine orange-mediated photodamage to cultured cells

Photosensitization mediated by the lysosomotropic, weakly basic dye acridine orange (AO) was studied on cultured J‐774 cells. The phototoxicity was found to be potentiated by elevated oxygen tension and reduced at low oxygen tension. Moreover, cell cultures pre‐exposed to the singlet oxygen scavenger sodium azide showed pronounced protection against the loss of viability induced by AO and blue light. AO‐mediated photosensitization was neither increased by pre‐exposure of cell cultures to ferric chloride or the catalase‐inhibitor aminotriazole nor decreased by exposure to deferoxamine. These observations suggest that type II (singlet oxygen‐mediated) reactions predominate over type I reactions (radical‐mediated). A rapid and pronounced decrease in lysosomal cathepsin L activity (up to 60%) was observed after an initial 10 min irradiation, indicating the lysosomal compartment to be an early target. This irradiation time did not, however, result in any substantial loss of viability. Levels of cytosolic lactate dehydrogenase were unaffected even after 30 min irradiation, indicating that neither cytosol nor plasma membrane is a primary target of the AO‐mediated photodamage. Glutathione depletion by pre‐exposure to buthionine‐S, R‐sulfoximine (BSO) much enhanced the sensitivity of J‐774 cells to AO‐mediated photosensitization, indicating a protective role for thiolcontaining compounds against AO‐mediated photodamage.

Alloxan cytotoxicity involves lysosomal damage

The cytotoxic effects of alloxan are not understood in any great detail, although they are considered to involve reactions mediated by oxygen‐derived free radicals. These reactive species may form extra‐or intracellularly following alloxan reduction, and result in cell damage through a number of complex interactions with a variety of macromolecules. The purpose of the present study was to elucidate further the early intracellular effects of alloxan on a model system of macrophage‐like cells in culture. Addition of alloxan (15 mM), without reducing agents, to the medium surrounding the cells (phosphate‐buffered saline, PBS, 37°C, pH 7.4) resulted in rapid lysosomal damage (disappearance of the proton gradient over the membrane) followed by severe cellular degeneration (swelling and blebbing) and 50% cell death (trypan blue dye exclusion test) within fifty min. Cells pretreated with the γ‐glutamyl cysteine synthetase‐inhibiting agent BSO, to decrease levels of intracellular glutathione, showed enhanced sensitivity to alloxan. The results are interpreted as indicating the cytotoxicity to result from intracellular formation of superoxide radicals, hydrogen peroxide and hydroxyl radicals, the latter within secondary lysosomes containing trace amounts of reactive iron (inducing Fenton reactions). The ensuing lysosomal membrane damage may result in leakage of lysosomal hydrolases and further cellular degeneration.

Laser Doppler perfusion imaging of the radial forearm flap: a clinical study.

Laser Doppler perfusion imaging (LDPI) allows non-invasive assessment of blood flow in a predefined area of skin rather than at one single point. We have used LDPI to study the pattern of skin blood flow in the radial forearm flap before and after the flap has been raised. The data were collected from a consecutive series of 11 patients with cancer of the oral cavity or oropharynx in whom the radial forearm flap had been used during the reconstructive procedure. Reperfusion leads to an immediate hyperaemic response both in the flap and the surrounding skin. This hyperaemia remains for at least the first 30 minutes after reperfusion. The perfusion of the radial side of the forearm skin distal to the flap is significantly lower than that on the ulnar side after the skin island has been raised and the distal artery divided. We suggest that LDPI is useful for monitoring the perfusion of free skin flaps.

Effects of alloxan and reducing agents on macrophages in culture

The diabetogenic effect of the quinonoid compound alloxan is not understood in detail although it supposedly involves reactions mediated by alloxan and oxygen radicals. These reactive species may form extra‐ or intracellular‐ and cause cell damage through a variety of complex interactions with several macromolecules. The purpose of this study was to elucidate early (≤ 60 min) effects of alloxan and reducing agents (cysteine and ascorbic acid) on cultured macrophages, as assayed by the trypan blue dye exclusion test and the sensitive fluorescein diacetate and propidium iodide (FDA/PI) double staining technique. During the reactions between alloxan and reducing agents, oxygen was consumed as a sign of superoxide anion radical formation. When alloxan alone was added to two different culture media without serum, oxygen was still consumed, indicating formation of oxygen radicals due to the occurrence of reducing substances in cell culture media. This finding demonstrated the necessity of performing further studies in solutions without reducing capacity, e.g. in phosphate‐buffered saline. The experiments showed that exposure of normal and malignant macrophages to alloxan and reducing substances resulted in rapidly occurring plasma membrane damage and ensuing cell death. Separate addition of catalase, desferrioxamine or superoxide dismutase resulted in evident, slight and no protection, respectively. The combinations of (i) catalase and desferrioxamine, and (ii) catalase, desferrioxamine and superoxide dismutase, however, inhibited cell damage in a pronounced and complete way, respectively. The results are interpreted as indicating cell damage due to the extracellular formation of hydrogen peroxide and hydroxyl radicals. The latter in close proximity to the cells and acting on the plasma membrane, while the former, after diffusing into the cell, may have several intracellular targets. The FDA/PI technique proved its value as a quantifiable method for the evaluation not only of cell death but also of cell damage with computer‐based fluorometry.

Intestinal obstruction following harvest of VRAM-flap for reconstruction of a large perineal defect.

Abstract A patient with locally advanced adenocarcinoma of the rectum was operated with abdominoperineal resection and perineal reconstruction with a vertical rectus abdominis musculocutaneous flap. Six days postoperatively, there was herniation of the small bowel, between the anterior and posterior rectus sheaths, to a subcutaneous location.