New Findings in PiZZ alpha1-antitrypsin deficiency-related panniculitis. Demonstration of skin polymers and high dosing requirements of intravenous augmentation therapy.

Panniculitis is a recognized but unusual complication of a severe deficiency of alpha1-antitrypsin (AAT), with fewer than 100 cases described to date. Like the pathogenesis of emphysema in severe PiZZ deficiency of AAT, panniculitis has been hypothesized to be an inflammatory process, possibly related to Z AAT polymer formation and to an unopposed anti-inflammatory screen in the context of deficient serum levels of AAT. The current report presents a 31-year-old woman with PiZZ AAT deficiency-associated panniculitis. Our case extends current knowledge of AAT-associated panniculitis in 2 ways: (1) we demonstrate Z-type AAT polymers in the skin, which supports the inflammatory pathogenesis of panniculitis and the potential pro-inflammatory role of polymers; (2) we show that a high dose and long-term use of intravenous augmentation therapy (90 mg/kg body weight once weekly during 3 years) can ameliorate the frequency and severity of panniculitis associated with AAT deficiency.

A novel cell force sensor for quantification of traction during cell spreading and contact guidance.

In this work, we present a ridged, microfabricated, force sensor that can be used to investigate mechanical interactions between cells exhibiting contact guidance and the underlying cell culture substrate, and a proof-of-function evaluation of the force sensor performance. The substrates contain arrays of vertical pillars between solid ridges that were microfabricated in silicon wafers using photolithography and deep reactive ion etching. The spring constant of the pillars was measured by atomic force microscopy. For time-lapse experiments, cells were seeded on the pillared substrates and cultured in an on-stage incubator on a microscope equipped with reflected differential interference contrast optics. Endothelial cells (ECs) and fibroblasts were observed during attachment, spreading, and migration. Custom image analysis software was developed to resolve cell borders, cell alignment to the pillars and migration, displacements of individual pillars, and to quantify cell traction forces. Contact guidance classification was based on cell alignment and movement angles with respect to microfabricated ridges, as well as cell elongation. In initial investigations made with the ridged cell force sensor, we have observed contact guidance in ECs but not in fibroblast cells. A difference in maximal amplitude of mechanical forces was observed between a contact-guided and non-contact-guided, but mobile, EC. However, further experiments are required to determine the statistical significance of this observation. By chance, we observed another feature of cell behavior, namely a reversion of cell force direction. The direction of forces measured under rounded fibroblast cells changed from outwards during early cell attachment to inwards during further observation of the spreading phase. The range of forces measured under fibroblasts (up to 138 nN) was greater than that measured in EC (up to 57 nN), showing that the rigid silicon sensor is capable of resolving a large range of forces, and hence detection of differences in traction forces between cell types. These observations indicate proof-of-function of the ridged cell force sensor to induce contact guidance, and that the pillared cell force sensor constructed in rigid silicon has the necessary sensitivity to detect differences in traction force vectors between different cell phenotypes and morphologies.

COX-2 is necessary for venous ligation-mediated bone adaptation in mice.

In osteoblasts, cyclooxygenase 2 (COX-2) is the major isozyme responsible for production of prostaglandins. Prostaglandins are local mediators of bone resorption and formation and are known to be involved in bone's adaptive response to fluid shear stress (FSS). We have previously described a model of trabecular bone loss in hindlimb-suspended mice and rats and demonstrated partial protection from osteopenia by ligation of the femoral vein. The increased FSS resulting from this ligation drove bone adaptation in the absence of mechanical loading. In this study, we investigated the role of COX-2 in this adaptive response to FSS by use of COX-2 knockout mice. COX-2 knockout ("KO"), COX-2 heterozygote ("HET"), and COX-2 wild-type ("WT") animals all lost comparable amounts of trabecular bone from sham-operated limbs as a result of suspension. In WT mice, loss of trabecular BMD in the venous-ligated limb was significantly less than that of the sham-operated limb; this effect, however, was not seen in KO or HET mice. Percentage gain in femoral periosteal circumference was greater in the ligated limb than the sham-operated limb for WT mice. KO and HET mice already possess femora of larger periosteal circumference than their WT littermates and ligation in these bones did not result in an increase in perimeter relative to sham. Histomorphometry on embedded bones revealed thinner cortices and less mineralizing perimeter in KO femora than controls. In conclusion, this is the first in vivo study to show that fluid-flow-mediated bone adaptation, independent of mechanical strain, is COX-2 dependent.

Does mesh implantation affect the spermatic cord structures after inguinal hernia surgery? An experimental study in rats.

BACKGROUND: Inguinal hernia repair is the most common operation in general surgery. Prosthetic reinforcement of the inguinal area with polypropylene mesh has increased dramatically in the last decade. The aim of this study was to evaluate how different types of mesh affect the spermatic cord structures. METHODS: Thirty rats were divided into three groups. The spermatic cord was dissected free and a conventional suture repair was performed in group I, an operation mimicking the Lichtenstein operation with a heavyweight polypropylene mesh in group II and the same operation using large pore, lightweighted polypropylene/polyglactin composite mesh in group III. A vasography was performed after 90 days. The cross-sectional area of the vas deferens and s-testosterone from the spermatic vein were measured using the contralateral side as control. Light microscopy of the inguinal canal was performed and inflammation and fibrosis were graded. RESULTS: Vasography revealed patent vas deferens in all animals. In group III, there was a lower s-testosterone in the spermatic vein and a reduced cross-sectional area of the vas deferens on the operated compared to the control side. However, there was no difference in the other groups and there was no significant difference in s-testosterone levels between the groups. There was significantly more inflammation and fibrosis after mesh repair compared to suture repair, but there was no difference between the two mesh groups. Unexpectedly, polyglactin fibres were still seen in specimens in group III after 90 days. CONCLUSION: In conclusion, the only effect on the spermatic cord structures in a rat model is seen as an impaired s-testosterone production and a reduced cross-sectional area of the vas deferens after use of a low-weight composite mesh compared to the control side. No difference in inflammation or fibrosis was found between heavyweight polypropylene mesh and low-weight composite mesh.

Reactive capsule formation around soft-tissue implants is related to cell necrosis.

Low-density polethylene disks with smooth or course surfaces were implanted in the abdominal wall of rats, and the tissue response was evaluated after 1, 6, or 12 weeks. Cell damage was detected by two different methods. Cells with increased membrane permeability could be identified using fluorescence microscopy by injection of propidium iodide prior to the killing of the rats. Second, cell death was verified by detection of DNA fragmentation. At 1 week a considerable number of the interfacial cells was stained with propidium iodide. Propidium-iodide-positive cells also were enriched at the edges of the disks irrespective of surface texture. The numbers of positive interfacial cells decreased markedly over time. Cells with DNA fragmentation initially displayed a scattered distribution; at later time points they appeared mainly in the outer portion of the enveloping capsule. The reactive capsule was thicker for the smooth surface, and there was a positive correlation between capsule thickness and propidium-iodide-positive cells at earlier implantation periods. The results suggest that the thickness of the reactive capsule is related to the extent of cell necrosis. It is suggested that the major initiator for this cell necrosis is mechanical shear since cell necrosis was found mainly in areas where mechanical shear could be expected.

Tissue reactions to polyethylene implants with different surface topography.

This study investigates the importance of implant surface topography on soft tissue response. The tissue response in the rat abdominal wall to discs of low density polyethylene with smooth to coarse surfaces was evaluated after one, six or 12 weeks. Capsule thickness and immunohistochemical quantification of monocytes-macrophages were used as measures. The macrophage specific antibody ED1 was used for identification of newly recruited macrophages and the ED2 antibody for the mature tissue macrophages. The smoother surfaces gave a thicker capsule than the rougher surfaces, and at one week also larger total numbers of cells and ED1 positive macrophages at interface. The capsule thickness increased over time for the smooth and intermediate surface topographies. In contrast, the cell numbers generally decreased over time. In conclusion, a coarse surface elicited lesser tissue reaction compared with a smooth surface.

Anti-inflammatory properties of titanium in the joint environment. An experimental study in rats.

Little is known about the tissue reactions to various implant materials which coincide with an inflammatory reaction. We used the avridine arthritis rat model to evaluate the tissue response in the synovial, interstitial and subcutaneous tissues after implant insertion. Quantitative immunohistochemistry showed that normal joint synovial tissue is dominated by ED2-positive resident macrophages. Polyethylene implants induced a much stronger foreign-body reaction than titanium implants, as measured by the number of interfacial ED1-positive macrophages. The tissue response to titanium and polyethylene was also vastly different in arthritic synovial tissue compared with control tissue. It is likely that these biomaterials interact differently with inflammatory cells or intermediary compounds. It may be that arthritic synovial tissue produces reactive oxygen intermediates (free radicals) with which titanium has a unique anti-inflammatory interaction in vitro.

Analysis of the inflammatory response to titanium and PTFE implants in soft tissue by macrophage phenotype quantification.

Implants of commercially pure titanium and polytetrafluoroethylene (PTFE) were inserted in the rat abdominal wall for 1, 6 or 12 wk. The foreign body reaction was evaluated by immunohistochemical quantification of monocytes/macrophages and by the thickness of the foreign-body capsule. At all time intervals, the majority of interfacial cells were ED1-positive while ED2-positive cells were localized deeper in the tissue. Neither titanium nor PTFE displayed a significant change in capsule thickness over time. The total cell numbers decreased overtime for both types of material. At 12 wk the PTFE implants, compared to titanium, were surrounded by a significantly thicker reactive capsule with larger total cell numbers. No significant differences were seen in the macrophage subset response between the two types of implants. Thus, the present study showed differences between titanium and PTFE at 12 wk but not at earlier time points.

Nerve regeneration in a 'pseudo-nerve' graft created in a silicone tube.

The pseudo-nerve, which contains longitudinal Schwann cell columns without axons and surrounded by perineurium-like tissue but no axons (Q. Zhao, L.B. Dahlin, M. Kanje, G. Lundborg, Brain Res. 592 (1992) 106-114), was applied as a graft to repair nerve defect in rats. Creation of the pseudo-nerve was accomplished by inserting the proximal and distal stumps of a cut sciatic nerve into a silicone tube. The proximal insert was cut far proximally to prevent axons from entering the tube. After 4 weeks, the pseudo-nerve was harvested, trimmed into a 10-mm long graft and transplanted into a corresponding defect of the contralateral sciatic nerve. Nerve regeneration through the pseudo-nerve was examined by pinch reflex test and neurofilament staining after 6 days or by morphology after 4, 6 or 8 weeks. The results showed that the pseudo-nerve could induce nerve regeneration to a similar extend as a real nerve graft. The neurobiological composition of the pseudo-nerve and the factors influencing its formation were also studied. By double staining of S-100 and laminin we found that the longitudinally organized Schwann cell columns in the pseudo-nerve were surrounded by basal laminae and ensheathed by a layer of vascularized perineurium-like tissue. Macrophages (ED1 and ED2) and their products interleukin-1beta (IL-1beta) and transforming growth factor-beta1 (TGF-beta1) were constantly present in the pseudo-nerve. Besides, the size of tube was a crucial factor in influencing pseudo-nerve formation, e.g. a thicker pseudo-nerve was formed in tubes with larger diameters or shorter gap lengths. No pseudo-nerve was formed when the gap was 15 mm long. When both proximal and distal inserts were isolated nerve segments the pseudo-nerve was still formed but thin, probably because of compromised vascular supply. Taken together, the results suggested that the pseudo-nerve contains the essential neurobiological elements to induce successful axonal elongation.

Leukocyte contribution to parenchymal cell death in an experimental model of inflammation.

The relationship between leukocyte migration and parenchymal cell death in vivo remains poorly documented. Accordingly, cell killing in the rat mesentery, as recorded by propidium iodide staining, was investigated with an intravital approach. Superfusion of platelet-activating factor (PAF, 10(-8) M) or N-formyl-methionyl-leucyl-phenylalanine (fMLP, 10(-8) M) led to extensive leukocyte extravasation but no significant cell death. In contrast, pretreatment with 10(-8) M PAF or fMLP for 1 h, followed by superfusion of PAF in combination with fMLP (both at 10(-8) M) led to an increase in cell death. Mesenteric parenchymal cells but no endothelial cells were killed. Some of the dead cells were identified as granulocytes/monocytes that were already in the tissue at the start of the experiment. The incidence of cell death was lower but not eliminated when leukocyte migration was blocked with a monoclonal antibody against CD18. A xanthine oxidase inhibitor, BOF-4272, failed to diminish cell death, whereas a hydroxyl radical scavenger, dimethylthiourea, attenuated cell killing without an effect on the number of adhering and migrating leukocytes. These observations demonstrate that leukocytes serve as a factor in the killing of extravascular cells only after the development of a level of stimulation that differs from that required to induce a migratory stimulus into the extravascular space.

Inflammatory reaction dependence on implant localization in rat soft tissue models.

This study compares two different implantation models in soft tissue in rat abdominal wall with regard to inflammatory reactions. Titanium rods and discs, penetrating or not penetrating the peritoneal wall respectively, were implanted. After 3, 10 or 30 days the distribution of monocytes/macrophages and cytokines (interleukin-1 and transforming growth factor-beta) in the tissue adjacent to the implants was investigated under immunohistochemistry. The macrophage-specific antibody, ED1, was used for the identification of newly recruited macrophages and the ED2 antibody was used for the mature tissue macrophages. After 10 days the non-penetrating implants had a larger number of cells close to the implant than the penetrating implants. The opposite was seen after 30 days implantation, with a larger number of cells around the penetrating implants. At all time intervals the penetrating implants had a thicker reactive capsule. The cytokines interleukin-1beta and transforming growth factor-beta could be detected in the reactive tissue adjacent to both types of implants, without obvious differences for the two implant situations. The biocompatibility of a material appears to be influenced by the localization of the implant. In addition, it seems to be of importance to extend the follow-up periods further, as we cannot assume that steady state is reached at 30 days implantation.

The role of macrophages in bioartificial nerve grafts based on resorbable guiding filament structures.

A 10 mm gap in a rat sciatic nerve was bridged by a bioartificial nerve graft consisting of a silicone tube containing seven longitudinally placed filaments made of non-resorbable material (polyamide [Ethilon]) or resorbable materials (polydioxanon [PDS], polyglactin [Vicryl] or catgut). The purpose was to study the tissue reaction induced by the four different types of materials. At 4 weeks an immunocytochemical technique, using ED1 and ED2 monoclonal antibodies, was used to study the presence and location of macrophages. A large number of macrophages were found accumulating on the surface of catgut and polyglactin, while few were found on the surface of polyamide and polydioxanon filaments. It is concluded that the cell layers on the filament surface mainly consisted of ED1 positive cells and their thickness depends on the filament materials.

Bioartificial nerve grafts based on absorbable guiding filament structures--early observations.

Gaps 10 mm wide in the sciatic nerves of 64 rats were bridged by bioartificial nerve grafts consisting of a silicone tube containing seven longitudinally placed filaments made of non-absorbable, (polyamide [Ethilon]) or absorbable, material (polydioxanone [PDS], polyglactin [Vicryl], and catgut). The purpose was to study the organisation of axonal growth inside the tube along such filaments. After two and four weeks histological techniques were used to study the contents of the tube and at four weeks immunohistological techniques were used to confirm the presence of axons distal to the tube. In all experimental groups axons had traversed the tube and reached the distal segment after four weeks. Inside the tube axons were organised in multiple minifascicles in all groups, but there were no axons growing in direct contact with the filaments. We conclude that resorbable filaments placed inside a silicone tube do not disturb axonal growth across the tube.

Rat sciatic nerve regeneration through a micromachined silicon chip.

The capacity of regenerating nerve fibres to grow through a perforated silicon chip was tested using the silicone chamber model for nerve regeneration. The chips were fabricated as circular membranes, 4 mm in diameter, thickness 60 microns, with a perforated area, 2 mm in diameter, in the centre. Three types of chips were fabricated utilizing anisotropic etching. The chips were glued with silicone adhesive between two halves of silicone rubber tubing (total length 8 mm, inner diameter 1.8 mm, outer diameter 3.0 mm) which was used to bridge a 4 mm gap between the proximal and distal nerve stumps of a transected rat sciatic nerve. The capacity of regenerating nerve fibres to grow through the holes of the chip was analysed by light and scanning electron microscopy after 4 or 16 weeks of regeneration. Furthermore, the muscle contractility force of the gastrocnemius muscle was measured after 16 weeks of regeneration and compared as a percentage of the contralateral uninjured side. Nerves generated through chips with hole diameters of 10 or 50 microns were morphological and functional failures. The nerve structures distal to chips with hole diameters of 100 microns contained many myelinated nerve fibres in a minifascicular pattern after both 4 and 16 weeks of regeneration. The muscle contractility force was 56% of that of contralateral control muscles.

Resorbable filament structures as a scaffold for matrix formation and axonal growth in bioartificial nerve grafts: long term observations.

Gaps, 10 mm wide, in rat sciatic nerves were bridged by bioartificial nerve grafts consisting of a silicone tube containing seven longitudinally placed synthetic filaments, which were expected to serve as a scaffold for axonal growth. The filaments were made of non-resorbable material (polyamide [Ethilon®]) or resorbable material (polydioxanon [PDS®], polyglactin [Vicryl®] or catgut). The purpose was to study the influence of resorbable materials on axonal regeneration and to choose, in the long term, the best filament material among the four. After 3 and 6 months, histological techniques were used to study the regenerated nerve structure. The total axon number in the nerve segment distal to the silicone chamber was counted in all specimens at 6 months. The histological findings were different depending on the filament materials; all the three resorbable materials showing significantly larger numbers of axons than polyamide (non-resorbable). All materials were covered with several layers of more or less flattened cells. These results indicate that resorbable filaments are superior to non-resorbable filaments when used as a scaffold inside a silicone tube, and polyglactin seems ideal for this purpose.

Tissue response to commercial silicone and polyurethane elastomers after different sterilization procedures.

Two different commercial polymeric materials, a silicone and a polyurethane (PUR), were studied with regard to correlations between the chemical and physical compositions of the polymer surfaces and the biological response on implantation. Test specimens of the materials were manufactured according to standard procedures. The specimens were implanted in rats for 10 and 90 days. Before implantation the polymers were sterilized in three different ways, namely, beta irradiation, ethylene oxide sterilization and steam sterilization. The polymers were characterized before and after the implantation with respect to the chemical composition and the morphology of the surfaces. After implantation the biological response was evaluated by counting numbers of macrophages, giant cells, fibroblasts and other cells present at the surfaces. The thickness of the fibrous capsule surrounding the test specimens was measured at the thickest and thinnest parts. PUR surfaces showed signs of degradation already after sterilization and after 10 to 90 days of implantation, pits and cracks appeared, especially in the ethylene oxide sterilized samples. However, differences in the biological responses were small and independent of the sterilization method. After 10 days of implantation the capsule thickness and the amounts of cell material adhering at the surfaces were different, and it appears that the silicone rubber induces more tissue response than PUR. The differences in the early tissue response evened out after 90 days implantation time and a steady state situation evolved, which was similar for the silicone and the polyurethane.

Effects of E-selectin and P-selectin blockade on neutrophil sequestration in tissues and neutrophil oxidative burst in burned rats.

OBJECTIVE: Neutrophil deposition in tissues (leukosequestration) after shock may produce local tissue injury from proteases and high-energy oxygen species released from sequestered neutrophils. The initial step in the binding of neutrophils to capillary endothelium is the interaction of adhesion molecule (selectin) receptors between neutrophils and endothelial cells. We quantified leukosequestration in the tissues of burned rats using two methods of analysis: a) measurement of lung myeloperoxidase; and b) measurement of radiolabeled neutrophils and erythrocytes deposited in multiple tissues. We then determined the ability of a selectin receptor blocking agent to affect neutrophil deposition in tissues after burn injury. DESIGN: Prospective, controlled, laboratory study. SETTING: University research laboratory. SUBJECTS: Male Wistar rats (200 to 300 g). INTERVENTIONS: After tracheostomy and venous cannulation, rats received 17% total body surface area full-thickness contact burns and were resuscitated with saline (20 mL i.p.). Experimental animals received 2 mg/kg body weight i.v. administration of a P- and E-selectin blocking monoclonal antibody, CY-1747, immediately after burn. Lung tissue neutrophils were estimated by measuring myeloperoxidase in lung tissue. Neutrophil retention in lung, liver, spleen, gut, skin, muscle, kidney, and brain tissues was determined by removing (preburn) and differentially radiolabeling neutrophils (111In) and erythrocytes (51Cr), reinfusing cells 4.5 hrs after burn, and measuring tissue radioactivity 30 mins later. Edema was estimated by measuring extravasated 125 I-labeled albumin in the various tissues. Peripheral blood neutrophils were analyzed for intracellular hydrogen peroxide content, utilizing a fluorescent dye that reacts with hydrogen peroxide, coupled with analysis of cell fluorescence by flow cytometry. MEASUREMENTS AND MAIN RESULTS: Myeloperoxidase concentration was increased in lungs 5 hrs after burn (p < .05), indicating neutrophil deposition. Radioisotope studies demonstrated significant (p < .05) leukosequestration into the lung, gut, kidney, skin, and brain tissues at 5 hrs after burn. Flow cytometry showed increased intracellular hydrogen peroxide content in peripheral blood neutrophils 5 hrs after burn. Tissue edema, manifested by radiolabeled albumin retention, was not seen in any tissues. Postburn neutrophil deposition in lungs and liver was blocked (p < .05) by administration of CY-1747 after burn, but maximal neutrophil hydrogen peroxide content was unaffected. CONCLUSION: Burn injury in rats results in accumulation of neutrophils in multiple tissues. Neutrophil deposition in the lungs and liver is blocked by administration of the E/P-selectin blocking antibody, CY-1747. Since sequestration of metabolically active neutrophils may induce tissue injury, therapies that block postburn leukosequestration may improve clinical outcomes by limiting remote tissue injury.

Effects of recombinant bactericidal/permeability-increasing protein (rBPI23) on neutrophil activity in burned rats.

Bactericidal/permeability-increasing protein (BPI) is a neutrophil granule protein with potent bactericidal and lipopolysaccharide (LPS)-neutralizing activities. The purpose of this study was to determine if a human recombinant BPI product, rBPI23, would influence neutrophil (PMN) sequestration into various tissues in a rat burn injury model. Leukosequestration may produce local tissue injury from proteases and high-energy oxygen species released from PMNs. Rats received tracheostomy and venous cannulation, then received 17 to 20% total body surface area full-thickness contact burns and resuscitation with 20 ml, of intraperitoneal saline. Ten mg/kg body weight rBPI23 in saline was given by intravenous injection immediately after burn injury, followed by intravenous doses of 2 mg/kg at 2 and 4 hours. Control animals received intravenous saline only. PMN retention in lung, liver, spleen, gut, skin, muscle, kidney, and brain tissues was determined by removing (before burn injury) and differentially radiolabeling PMNs (111In) and erythrocytes (51Cr), reinfusing cells 4.5 hours after burn injury, and measuring tissue radioactivity 30 minutes later. Edema was estimated by measuring extravasated 125I-labeled albumin in the various tissues, 30 minutes after injection. Peripheral blood PMNS were analyzed for intracellular H2O2 content by flow cytometry using a fluorescent dye that reacts with H2O2. Radioisotope studies demonstrated significant (p < 0.05) leukosequestration into lung, liver, gut, kidney, and skin tissues at 5 hours after burn injury. Tissue edema, manifested by radiolabeled albumin retention, was not observed in any tissues. Postburn PMN deposition in lungs and skin was decreased (p < 0.05) by the immediate administration of rBPI23 after burn injury. Flow cytometry showed increased intracellular H2O2 content in peripheral blood PMNs 5 hours after burn injury (p < 0.05), which was unaffected by administration of rBPI23. Since sequestration of metabolically active PMNs may induce tissue injury, therapies that block leukosequestration after burn injury may improve clinical outcomes by limiting remote tissue injury.

Neutrophil activation and tissue neutrophil sequestration in a rat model of thermal injury.

Neutrophil (PMN) deposition in tissues (leukosequestration) after shock may produce local tissue injury from proteases and oxygen intermediaries which are released from sequestered PMNs. We quantified leukosequestration in tissues in burned rats using two methods of analysis: 1), measurement of lung myeloperoxidase (MPO); 2), measurement of radiolabeled PMNs and erythrocytes deposited in multiple tissues. After tracheostomy and venous cannulation, rats received 17% TBSA full-thickness contact burns and were resuscitated with 20 cc intraperitoneal saline. Lung PMNs were estimated by measuring MPO in lung tissue. PMN influx into lung, liver, spleen, gut, skin, muscle, kidney, and brain was determined by removing (preburn) and differentially radiolabeling PMNs (111In) and erythrocytes (51Cr), reinfusing cells 4.5 hr postburn, and measuring tissue radioactivity 5 hr postburn. Tissue edema was measured by determining extravasation of 125I-labeled albumin in tissues. Peripheral blood PMNs were analyzed for intracellular H2O2 content utilizing a fluorescent dye which reacts with H2O2 coupled with analysis of cell fluorescence by flow cytometry. MPO was elevated in lungs 8 hr postburn (P < 0.05). PMN influx into lung tissues was confirmed by histologic examination. Radioisotope studies demonstrated significant (P < 0.05) leukosequestration into lung, gut, kidney, skin, and brain tissues at 5 hr postburn. Respiratory burst activity of peripheral blood PMNs was increased 5 hr postburn (P < 0.05). Flow cytometric analysis indicated that peripheral blood PMNs were capable of producing markedly increased H2O2 levels 5 hr postburn. Tissue edema, manifested by radiolabeled albumin influx, was not seen in any tissues. Since others have shown that sequestration of metabolically active PMNs may induce remote tissue injury, therapies which block postburn leukosequestration may be able to improve clinical outcomes by limiting remote tissue injury.

Immunohistochemical study of the soft tissue around long-term skin-penetrating titanium implants.

Bone-anchored percutaneous titanium implants have become a well-established clinical procedure with a low incidence of adverse reactions. However, passage through the skin leads to a breach in the barrier to exogenous pathogens. In the present study, monoclonal antibodies were used to investigate the distribution of lymphocyte subpopulations in the soft tissue around such implants. Eight biopsies from patients with clinically irritated skin, five from non-irritated and eight from skin without skin-penetrating implants were analysed. The number of immune cells was increased in the group of patients with skin penetration compared with patients without skin penetration. In the group with clinical irritation there was an increased level of B-lymphocytes compared with those without irritation. The data suggest that there is an immunological compensation for the mechanical loss in barrier function at these implants and that an antibody-mediated response is present at clinical signs of irritation.