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Biomedical subjects

W J Lindblad

Publications and source records attributed to W J Lindblad.

At least 19 recordsLinked to original sources
PubMed1996

Pneumothorax and wound dehiscence related to collagenase deregulation: treatment with diphenylhydantoin.

BACKGROUND: Wound dehiscence is an uncommon complication of operation, usually related to a recognized risk factor. A clinical dilemma arises when dehiscence has no identifiable cause or treatment. METHODS: We describe the case of a previously healthy 45-year-old man in whom recurrent spontaneous pneumothoraces developed followed by multiple dehiscences of thoracotomy, diaphragmatic, and abdominal wounds. Analysis over several years of laboratory investigation of cultured tissue from test incisions was initially unsuccessful. The patient was supported symptomatically until a remarkable laboratory finding enabled us to develop an effective treatment plan. RESULTS: Cultured patient fibroblasts were ultimately found to express abnormally elevated levels of collagenase, which could be inhibited by diphenylhydantoin (phenytoin) in vitro. Treatment of the patient with a course of diphenylhydantoin allowed adequate healing of test incisions and subsequent definitive surgical treatment with successful wound healing. CONCLUSIONS: This report of the rigorous application of the scientific method to the investigation and treatment of an enigmatic case of wound dehiscence might serve as a guide to surgeons faced with similar healing problems.

Abdomen↗
PubMed1992

Effect of age on wound healing in healthy human beings.

BACKGROUND: Although the elderly clinically have a higher rate of wound complications, the physiologic effect of age on wound healing in human beings is unknown. METHODS: Healthy young (18 to 55 years of age) and elderly (more than 65 years of age) human volunteers had a 2 x 2 cm, superficial, split-thickness wound created on the anterior aspect of the thigh, and the rate of epithelialization was assessed. For studies of fibroplasia, similar groups underwent subcutaneous implantation of polytetrafluoroethylene catheters, which were removed after 14 days. Biochemical analyses of the catheters included determinations of hydroxyproline, total alpha-amino nitrogen, and DNA. RESULTS: The elderly volunteers had a significant delay of 1.9 days in epithelialization. Analyses of the subcutaneously implanted catheters showed no difference in DNA content or hydroxyproline-accumulation; however, the young volunteers had a significantly higher amount of total alpha-amino nitrogen. CONCLUSIONS: In healthy humans, aging leads to delayed epithelialization. No effect of age on collagen synthesis was noted, although accumulation of wound noncollagenous protein was decreased. This decrease may impair the mechanical properties of scarring in aged human beings.

Adolescent↗
PubMed1991

Hepatocellular phenotype in vitro is influenced by biophysical features of the collagenous substratum.

Hepatocytes maintained on different substrata in vitro possess strikingly different morphological and biochemical features. Rounded, multicellular aggregates of hepatocytes are seen if the cells are plated onto Matrigel, a reconstituted basement membrane, whereas a flattened, monolayer of hepatocytes is observed with Vitrogen. Hepatocellular protein synthesis is much greater on the Matrigel, although collagen biosynthesis appears selectively enhanced on Vitrogen-grown hepatocytes. We determined that denatured type I collagen could be substituted for Matrigel as the substratum, with the hepatocytes remaining the same both morphologically and biochemically. This suggested that the cells respond to the biophysical state of the extracellular matrix not only to protein sequences that determine a binding site. Measurement of steady-state messenger RNA levels within cells cultured onto different matrices indicated that the fluid substrate of either Matrigel or denatured type I collagen were facilitative for induction of cytochrome P-450b/e, which was not seen with the rigid type I collagen substrata. In contrast the messenger RNA level for the cytoskeletal protein actin was decreased on the fluid matrices, suggesting that the rounded cells had a lower requirement for this protein. These findings indicate that hepatocytes are responsive to the biophysical state of the extracellular matrix, which can lead to significant changes in gene expression by the cells.

Actins↗
PubMed1990

Production of collagen type I by mouse peritoneal macrophages.

The origin and identity of the cells that accumulate and produce collagen in wound healing and in granulomatous and fibrotic processes have long remained unsettled. In this study, it was found that pure cultures of peritoneal macrophages produced hydroxy-L-proline not associated with Clq and produced detectable quantities of collagenase-digestible protein. Cultured macrophages synthesized collagen while retaining their phagocytic capacity. Immuno-blot analysis identified the collagen as Type I. This shows that macrophages have collagen-forming capacity and may be productive cells in pathologic collagen deposition.

Animals↗
PubMed1989

Protamine selectively inhibits collagen synthesis by human intestinal smooth muscle cells and other mesenchymal cells.

Collagen synthesis is a major function of human intestinal smooth muscle (HISM) cells and contributes to intestinal fibrosis in chronic inflammatory bowel disease. As an extension of previous in vitro studies of the role of heparin in regulating HISM cell proliferation and collagen synthesis, the effect of protamine sulfate was studied. Protamine decreased collagen production by 50% in confluent and proliferating cultures. This effect was concentration-dependent and was selective for collagen in that neither noncollagen production nor DNA accumulation in the culture plates was affected. Other human mesenchymal cells which produce collagen, such as dermal fibroblasts and aortic smooth muscle cells, responded to protamine in a similar fashion. Protamine has a strong cationic charge and is rich in lysine and arginine. To determine which of these properties was important in decreasing collagen production, the effect of protamine was compared to that of other polyionic compounds. Poly-L-lysine decreased collagen production to a lesser degree than protamine. Poly-L-arginine was toxic to the cells. Poly-L-glutamic acid, which has an opposite charge to protamine, had no effect. These findings suggest that both the number and the arrangement of lysyl residues, in addition to positive charge, are important. Binding assays demonstrated that protamine did not inhibit collagen production by binding to ascorbate in the culture medium. Electrophoretic separation and chromatography of collagen types expressed following protamine treatment showed that the ratio of type I to type III collagen remained 2:1. This observation suggests that suppression of collagen production is not specific to a particular collagen type. The selective inhibition of collagen production by protamine provides an important tool to study the regulation of collagen production in human cells and may also provide potential therapy of fibrotic disorders.

Ascorbic Acid↗
PubMed1989

Analysis of fibrogenic processes in denervated tissues of spinal cord injury patients.

To test the hypothesis that altered collagen metabolism is a contributing factor in the apparent delayed wound healing in denervated regions of spinal cord injury (SCI) patients, a tissue implant (PVA) was used to directly measure collagen deposition. Sterile PVA implants were placed subcutaneously in the inner aspect of the upper arm above the cord injury (innervated) and in the inner aspect of the upper leg below the cord injury (denervated) of 20 spinal cord injury patients and compared to eight healthy volunteers. On day 14, the implants were removed and analyzed histologically by trichrome stain and biochemically for hydroxyproline as a measure of collagen deposition. No remarkable histologic differences were observed in the sponge material removed from the upper regions compared to the lower denervated regions of the spinal cord injury patients. Sponges from both areas were infiltrated with fibroblasts containing well-developed rough endoplasmic reticulum and large quantities of trichrome-positive collagen. Likewise, upper and lower histology of controls was identical and nondistinguishable from the corresponding sections obtained from the spinal cord injury patients. Quantitation of the hydroxyproline in the arms of the spinal cord injury patients (n = 20) showed 4.3 +/- 0.7 nmol hydroxyproline per milligram of sponge compared to 4.1 +/- 0.4 nmol/mg in the denervated regions of the lower limb. The hydroxyproline content in the arms of control volunteers was 5.2 +/- 0.7 nmol/mg compared to 3.9 +/- 0.8 nmol/mg in the leg (n = 8). These observations suggest that fibrogenic processes in denervated regions are not reduced significantly compared to innervated regions.

Adult↗
PubMed1988

Collagen content and types in the intestinal strictures of Crohn's disease.

The collagen content and the relative amount of collagen types were quantitated in control intestine as well as in both inflamed and strictured intestine resected from patients with Crohn's disease. The major collagen type in control intestine was type I (68%), followed by types III (20%) and V (12%). In strictured intestine both collagen content and the relative amount of type V collagen were significantly increased compared with control intestine. Histologic studies demonstrated that in strictured specimens there was a striking proliferation of smooth muscle cells of the muscularis mucosae associated with an accumulation of collagen in the submucosa. The thickness of the muscularis propria was also increased. Immunohistochemical studies demonstrated small amounts of type V collagen in the submucosa of control bowel. In contrast, large amounts of type V collagen were seen in the fibrotic, expanded submucosa of strictured bowel, particularly in the areas where smooth muscle cells of the muscularis mucosae had proliferated. Intestinal strictures in Crohn's disease are therefore characterized by an accumulation of collagen, a proliferation of smooth muscle cells, and an increase in type V collagen, a collagen type produced in relatively large amounts by smooth muscle cells. These changes appear to result in both a loss of the normal compliance of the intestine and a thickening of the intestine wall, resulting ultimately in the intestinal obstruction so frequently seen in patients with Crohn's disease.

Amino Acids↗
PubMed1987

Induction of prolyl hydroxylase activity in a nonadherent population of human leukocytes.

A nonadherent population of human monocytes has been shown to express the collagen hydroxylating enzyme prolyl hydroxylase in vitro. Enzyme levels present in freshly isolated nonadherent cells were induced 300% during the first 72 hours of culturing, which could be suppressed by cycloheximide. Maximum induction required both a feeder layer of adherent leukocytes, and 10-15% autologous plasma. Biosynthesis of Clq, a protein which also is hydroxylated by prolyl hydroxylase, by the nonadherent cells was significantly less than the adherent monocytes. Therefore, this collagen biosynthetic marker enzyme was not associated with Clq synthesis, which suggests that the enzyme is present for collagen biosynthesis.

Cells, Cultured↗
PubMed1987

Collection of leukocytes, fibroblasts, and collagen within an implantable reservoir tube during tissue repair.

An implantable chamber consisting of a small reservoir and a perforated segment of silicone tubing has been developed for the collection of leukocytes, fibroblasts, and collagen to analyze inflammatory components and fibroplasia during tissue repair. Using aseptic techniques, these sterile chambers were placed into subcutaneous pockets on the backs of Sprague-Dawley rats with cells obtained in daily aspirates for 14 d. Differential cell counts were made by using aliquots from the wound fluid. The aspirated cells represented the characteristic, sequential influx of neutrophils, inflammatory macrophages, lymphocytes, activated macrophages, and fibroblasts documented in other models of tissue repair. On d 14, the connective tissue within the lumen of the silicone tube was removed and analyzed for collagen synthesis by measuring 3H-proline incorporation into collagenase-sensitive protein. This new device for studying wound healing provides a convenient means to harvest cells, fluid, and tissue for cellular, humoral, and biochemical analyses of tissue repair.

Animals↗
PubMed1987

Establishment of an adult rat fibroblast cell line for studies of collagenase regulation.

Several dermal fibroblast lines have been established from explants taken from adult rats. The cells have been cultured for 2 yr and possess stable and well-defined growth characteristics through subculture 18. The cells are readily stored in liquid nitrogen with good viability after thawing. Collagenase activity secreted into the culture medium of the cells at different periods of growth has been examined. There is an 88% drop in total enzyme activity present in the medium between 4 and 14 d of culture, when the cells were plated to reach confluence at Day 8 to 10. A more pronounced fall is noted at earlier times when the cells are plated at a higher density. The correlation between DNA content of the cell monolayer and enzyme activity was -0.895, indicating a possible relationship between the growth of cells and collagenase release.

Animals↗
PubMed1987

Fetal response to injury in the rabbit.

Fetal, neonatal, and adult tissue response to a standardized injury was studied using subcutaneous wound implants, linear incisions, and punch wounds in New Zealand white rabbits. In the fetus, sutured incisions healed by primary intention without antecedent inflammation. However, neither contraction nor healing by secondary intention was seen in punch or unsutured wounds. Healing both by primary and secondary intention following inflammatory infiltration was observed uniformly in neonatal and adult rabbits. Wound implants were extensively infiltrated with collagen in the adults studied; however, no collagen was seen in fetal implants and collagen hydroxyproline content could not even be detected by high performance liquid chromatography techniques; rather, a matrix rich in hyaluronic acid was found. The fetal tissue response to injury differs from the adult, proceeding in the absence of a classical inflammatory stimulus and lacking contractile capabilities. The deposition of extracellular matrix rich in hyaluronic acid but devoid of collagen suggests that the fetal response to injury may be a process more closely resembling regeneration or growth rather than repair by scar deposition.

Animals↗
PubMed1986

A subcutaneous implant for wound healing studies in humans.

A method for studying wound healing in humans is described. The technique is based on the production of a standard subcutaneous injury during implantation of a retrievable sponge. The injury is produced by introducing a small device (PVA implant) which consists of a 5.7-cm piece of perforated silicone tubing containing two pieces of polyvinyl alcohol sponge. The sponge provides a site for attracting inflammatory cells with subsequent fibroblast infiltration. The sterile PVA implant is inserted subcutaneously in the upper arm by means of a 12-gauge needle and remains there for 14 days. Upon removal, one sponge has hydroxyproline deposition quantitated using a high-performance liquid chromatography technique. The other sponge may be processed for light and electron microscopy or specialty staining. Other collagen determinations such as percentage neutral salt-soluble collagen are also possible. Using the PVA implant has made it possible to follow the kinetics of collagen deposition in the rat. There was a marked increase in collagen accumulation from Day 2 (0.89 nmole/mg sponge) to Day 14 (18 nmole/mg sponge) in the rat. Collagen deposition was also measured in human control subjects (5.07 nmole/mg sponge, n = 12) and compared to trauma patients (2.04 nmole/mg sponge, n = 5). Histologic staining showing fibroblast infiltration and collagen deposition correlated well with the biochemical findings. This implant, coupled with recent HPLC technology, provides a safe, acceptable technique to study human wound healing parameters and overcomes many of the limitations of previous methods.

Adult↗
PubMed1985

Cellular sources of fibrotic collagen.

Every somatic cell within vertebrate species contains genetic information and therefore the potential to synthesize collagen. Normally, when there is a requirement for new connective tissue following injury, cells of mesodermal origin are activated to meet that demand and supply sufficient amounts of collagen to return structure and function to the damaged tissue or organ. Under these conditions, nonconnective tissue cells either do not synthesize collagen or may produce only minute quantities which are barely detectable by our most sensitive techniques. However, when these nonmesodermal cells are exposed to an abnormal or toxic environment, collagen expression may become greatly enhanced. For example, when hepatocytes are removed from their normal in vivo environment and placed in cell culture, they demonstrate a dramatic increase in their potential to produce collagen. Hepatocytes may also express this ability to produce significant quantities of collagen when they are exposed to toxins or injury in vivo. The list of cells shown to produce collagen or contain enzymes related to collagen biosynthesis has expanded in recent years and now includes such diverse and specialized cells as glomerular mesangial, adipocytes, aortic endothelial, smooth muscle, Schwann cells, and lymphocytes. Therefore, the loss of structure and function in a tissue or organ due to fibrosis may result from excessive deposition of collagen from nonmesodermal sources as well as from connective tissue cells.

Animals↗
PubMed1984

Quantitation of hydroxyproline isomers in acid hydrolysates by high-performance liquid chromatography.

A method has been developed to rapidly separate and quantitate levels of hydroxy-L-proline isomers in tissue hydrolysates. The procedure incorporates derivatization of the imino acids with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole chloride followed by separation by high-performance liquid chromatography employing two C18 reverse-phase columns connected in series. Conditions for the derivatization procedure have been optimized for the selective reactivity of imino acids. The derivatized imino acid fractions are then quantitated spectrophotometrically at 495 nm. Using this technique, quantities above 40 pmol are readily detected for trans-4-hydroxyl-L-proline, trans-3-hydroxyl-L-proline, proline, and other imino acid analogs. The method is applicable to a wide range of clinical and experimental tissues.

4-Chloro-7-nitrobenzofurazan↗
PubMed1984

Glucocorticoids suppress formation of collagen by the hepatocyte. Studies in primary monolayer cultures of parenchymal cells prepared from adult rat liver.

We have established previously that the adult rat hepatocyte produces collagen under conditions of primary monolayer culture or under pathologic conditions in vivo. During the initial 3 days of incubation of hepatocytes in serum-free culture medium, collagen synthesis represented only 0.01%-0.05% of the synthesis of noncollagen proteins. Thereafter, rates of collagen synthesis rose dramatically, and after 9 days of incubation were increased an average tenfold in cultures derived from normal hepatocytes and 30-fold in cultures derived from regenerated liver. The spontaneous increase in the rates of collagen synthesis was almost completely blocked (94%) when cultures were incubated for 8 days in medium containing the synthetic glucocorticoid dexamethasone (10(-7) M). Dexamethasone was without effect on the synthesis of noncollagen proteins. Inhibition of collagen synthesis by dexamethasone exhibited a sigmoidal dose-response curve with 50% inhibition observed at a concentration of 10(-9) M. Collagen synthesis was diminished similarly by other glucocorticoids (hydrocortisone, methylprednisolone, triamcinolone, prednisone, all 10(-6) M) but not by other hormonal or nonhormonal steroids (progesterone, estradiol, testosterone, all 10(-5) M). We excluded increased collagen degradation as an explanation for these results by demonstrating that dexamethasone failed to increase the incorporation of radioactive proline into trichloroacetic acid-soluble radioactive hydroxyproline. Exposure of hepatocytes to dexamethasone during the first 48 h of incubation produced inhibition of collagen synthesis in 9-day-old cultures, even though washout studies with [3H]dexamethasone revealed negligible retention of the steroid by the cells. This observation suggests that among the initial, culture-adaptive responses of the hepatocyte are glucocorticoid-sensitive events that culminate in increased rates of collagen synthesis days later. We conclude that glucocorticoids may serve as natural suppressors of the potential of the hepatocyte to produce collagen.

Animals↗
PubMed1982

Mechanism for the differential induction of mutation by S9 activated benzo[a]pyrene employing either a glucose-6-phosphate-dependent NADPH-regenerating system or an isocitrate-dependent system.

A significant difference in mutation frequency has been observed in CHO cells exposed to benzo[a]pyrene with alternative activation systems. Each system employed rat-liver S9 homogenate with one using isocitrate dehydrogenase to provide reduced NADP, while the other method uses glucose-6-phosphate dehydrogenase. Total aryl hydrocarbon hydroxylase (AHH) activity was greater for the isocitrate dependent system, however, this yielded a lower level of HGPRT mutants. It was ascertained that this reduced mutation frequency may result from sequestering of B[a]P substrate by crystals in the medium, possibly calcium phosphate, which decreased the effective substrate concentration. This sequestration enhances B[a]P internalization, which would explain the dichotomy between the AHH values and the mutation frequency data. The production of specific B[a]P metabolites was also examined by reverse phase HPLC quantitation of extracts of solutions in which the two activation systems were used. The levels of 7,8 dihydroxybenzo[a]pyrene produced by the glucose-6-phosphate protocol were consistently greater than with isocitrate. This may also be a contributing mechanism for elevating the mutation frequency with this procedure. These results demonstrate several interactions between test compound, cells, and metabolizing system which must be considered with in vitro activation systems.

Animals↗