Peritoneal fine structure of inguinal hernia: a scanning electron microscope study

Mesothelial cells of the normal human peritoneum of the anterior abdominal wall are covered with numerous surface microvilli. These cells become partially denuded inside the sacs of direct and indirect inguinal hernias and so lose the protective property the microvillar covering may impart on them. These mesothelial cells of hernial sacs also acquire an extensive surface coat of fibrin-like material, presumably due to the loss of that protective property, which may as a result subject them to adhesions. There is a considerable collagen build-up in the subserosal fibrous tissue of sacs of both direct and indirect inguinal hernias. Such a build-up is at variance with the accepted current surgical concept which suggests a defect in collagen synthesis, rather than a build-up, as the cause of direct hernia.     

Alterations in copper and collagen metabolism in the Menkes syndrome and a new subtype of the Ehlers-Danlos syndrome

Cultured fibroblasts of 13 patients with the Menkes syndrome and two with a new subtype (type IX) of the Ehlers-Danlos syndrome (E-D IX patients) showed many very similar abnormalities in their copper and collagen metabolism. Both cell types had markedly increased copper concentrations and 64Cu incorporation, and this cation accumulated in metallothionein or a metallothionein-like protein, as previously established for Menkes cells. Histochemical staining indicated that copper was distributed diffusely throughout the cytoplasm in both cell types, this location being consistent with the accumulation in metallothionein. Both fibroblast types also had markedly low lysyl oxidase activity and distinctly increased extractability of newly synthesized collagen, whereas no abnormalities were present in cell viability, duplication rate, prolyl 4-hydroxylase activity, or collagen synthesis rate. A high negative correlation (P less than 0.001) was found in the pooled group of Menkes and E-D IX cells between cellular copper concentration (r = 0.804) or 64Cu incorporation (r = 0.863) and the logarithm of lysyl oxidase activity. There was also a high positive correlation (P less than 0.001) between cellular copper concentration and incorporation (r = 0.869). One of the two E-D IX patients was also shown to have similar changes in lysyl oxidase activity and collagen extractability in the skin biopsy specimen, suggesting that the abnormalities observed in cultured cells are similar to those present in vivo. The only distinct abnormality found in the cells of the parents of the E-D IX patients was an increased 64Cu incorporation in those of the mother, this finding being consistent with X-linked inheritance of the disorder.     

Lysyl oxidase and P-ATPase-7A expression during embryonic development in the rat

Lysyl oxidase activity is critical for the assembly and cross-linking of extracellular matrix proteins, such as collagen and elastin. Moreover, lysyl oxidase activity is sensitive to changes in copper status and genetic perturbations in copper transport, e.g., mutations in the P-type ATPase gene, ATP7A, associated with cellular copper transport. Lysyl oxidase may also serve as a vehicle for copper transport from extracellular matrix cells. Herein, we demonstrate that sufficient lysyl oxidase functional activity is present in the rat embryo at gestation day (GD) 9 to be detected in conventional enzyme assays. Estimation of embryonic lysyl oxidase functional activity, however, required partial purification in order to remove inhibitors. From GD 9 to GD 15, lysyl oxidase activity was relatively constant when expressed per unit of protein or DNA. In contrast, the steady-state levels of lysyl oxidase and ATP7A mRNA, measured by RT-PCR and expressed relative to total RNA and cyclophilin mRNA, increased approximately fourfold from GD 9 to 15. The pattern of temporal expression for ATP7A was consistent with its possible role in copper delivery to lysyl oxidase.     

Sliding indirect inguinal hernia

From 2 per cent to 5 per cent of all indirect inguinal hernias are of the sliding variety. (Sliding hernias are those in which part of the wall of the sac is formed by a viscus.) The proportion of sliding hernias is even higher in the aged. Hernias of this kind are found almost exclusively in males and usually on the left side. Preoperative diagnosis is not essential if the surgeon can recognize the lesion at operation and knows how to repair it properly. The LaRoque technique in which the peritoneal cavity is entered above the internal ring allows accurate definition of the pathological anatomy and effective repair of the hernia. It should be used in all true sliding indirect inguinal hernias.     

The large inguinal ring: its significance in the diagnosis of inguinal hernia

A large external inguinal ring is often reported by a medical examiner as a "potential hernia." This finding may cause the subject to be denied job opportunities and may make him apprehensive about many normal activities. The author believes that unless a sac is present and is causing symptoms that necessitate surgical relief, the term hernia should not be used, regardless of how it is qualified. The ordinary intraabdominal stresses due to coughing, sneezing, etc. increase intraabdominal tension more than heavy lifting, except with loads of nearly the body's own weight. The lifetime effect of such stresses can contribute to the development of a direct hernia, but most of these cannot be eliminated.     

How accurately can direct and indirect inguinal hernias be distinguished?

An inguinal hernia was clinically diagnosed as direct or indirect by paired surgeons of 134 occasions. When compared with the findings at operation the hernia was correctly diagnosed in 60 of 78 observations when it was indirect and in 33 of 56 when it was direct. The level of accuracy does not warrant continuing the practice of attempting to distinguish one type of inguinal hernia from another.     

Bilateral Hernias in the Female

An experience with 216 bilateral hernias in female patients is reviewed. The condition is rare, occurring only once in every 250 patients admitted for a hernia repair. Bilateral primary indirect inguinal hernias were the most frequent type. Bilateral primary femoral hernias were quite rare while bilateral primary direct inguinal hernias were even more uncommon. Other rare bilateral combinations are briefly described. The incidence in children is given.Etiological factors are discussed, emphasizing the strong posterior wall of the inguinal canal in females.Two per cent of patients developed a recurrent hernia; one per cent of hernias recurred. No recurrence following a bilateral primary indirect inguinal hernia repair and no “femoral” recurrence following inguinal repair were recorded.     

Type IX Ehlers-Danlos syndrome and Menkes syndrome: the decrease in lysyl oxidase activity is associated with a corresponding deficiency in the enzyme protein.

Type IX of the Ehlers-Danlos syndrome (E-D IX) and the Menkes syndrome are X-linked recessively inherited disorders characterized by abnormalities in copper metabolism. These abnormalities are associated with a severe reduction in the activity of lysyl oxidase, the extracellular copper enzyme that initiates crosslinking of collagens and elastin. No increase in this deficient enzyme activity was obtained when culture media from fibroblasts of patients with E-D IX or the Menkes syndrome were incubated with copper under various conditions in vitro. A distinct, although small, increase in lysyl oxidase activity was obtained, however, when copper-supplemented media were used during culturing of the fibroblasts, although even under these conditions, the enzyme activity in the media from the affected cells remained markedly below that of the controls. Immunoprecipitation, dot-blotting, and immunoperoxidase staining experiments with antisera to human lysyl oxidase indicated that fibroblasts from patients with E-D IX or the Menkes syndrome do not secrete into their medium, or contain inside the cell, any significant amounts of a copper-deficient, catalytically inactive lysyl oxidase protein. These findings appear to be consistent with the hypothesis that synthesis of the lysyl oxidase protein itself is impaired. The possibility is not excluded, however, that a copper-deficient enzyme protein may be synthesized in normal amounts but become degraded very rapidly inside the cell. The failure to obtain any large increase in the deficient lysyl oxidase activity upon various forms of copper administration suggests that it may not be possible to obtain any significant improvement in the connective tissue manifestations of these disorders by copper therapy.     

Changes in collagen cross-linking and lysyl oxidase by estrogen.

Dermal collagen solubility and lysyl oxidase activity of bones were measured in DDD mice of advancing age. Insoluble fractions of the dermal collagen increased more rapidly in females than in males after 5 weeks of age. Activity of the lysyl oxidase extracted from bones was higher in females than in males after 4 weeks of age. After sexual maturation, such sex differences were always observed in skin as well as in bone tissue. In other experimental animals, dermal collagen solubility was markedly decreased by estrogen treatment and lysyl oxidase was remarkably activated by estrogen in both skin and bone. Thus it is clear that estrogen stimulates the enzyme activity and accelerates the maturation of collagen and elastin in extracellular space.     

Multiple bone morphogenetic protein 1-related mammalian metalloproteinases process pro-lysyl oxidase at the correct physiological site and control lysyl oxidase activation in mouse embryo fibroblast cultures

Lysyl oxidase catalyzes the final enzymatic step required for collagen and elastin cross-linking in extracellular matrix biosynthesis. Pro-lysyl oxidase is processed by procollagen C-proteinase activity, which also removes the C-propeptides of procollagens I-III. The Bmp1 gene encodes two procollagen C-proteinases: bone morphogenetic protein 1 (BMP-1) and mammalian Tolloid (mTLD). Mammalian Tolloid-like (mTLL)-1 and -2 are two genetically distinct BMP-1-related proteinases, and mTLL-1 has been shown to have procollagen C-proteinase activity. The present study is the first to directly compare pro-lysyl oxidase processing by these four related proteinases. In vitro assays with purified recombinant enzymes show that all four proteinases productively cleave pro-lysyl oxidase at the correct physiological site but that BMP-1 is 3-, 15-, and 20-fold more efficient than mTLL-1, mTLL-2, and mTLD, respectively. To more directly assess the roles of BMP-1 and mTLL-1 in lysyl oxidase activation by connective tissue cells, fibroblasts cultured from Bmp1-null, Tll1-null, and Bmp1/Tll1 double null mouse embryos, thus lacking BMP-1/mTLD, mTLL-1, or all three enzymes, respectively, were assayed for lysyl oxidase enzyme activity and for accumulation of pro-lysyl oxidase and mature approximately 30-kDa lysyl oxidase. Wild type cells or cells singly null for Bmp1 or Tll1 all produced both pro-lysyl oxidase and processed lysyl oxidase at similar levels, indicating apparently normal levels of processing, consistent with enzyme activity data. In contrast, double null Bmp1/Tll1 cells produced predominantly unprocessed 50-kDa pro-lysyl oxidase and had lysyl oxidase enzyme activity diminished by 70% compared with wild type, Bmp1-null, and Tll1-null cells. Thus, the combination of BMP-1/mTLD and mTLL-1 is shown to be responsible for the majority of processing leading to activation of lysyl oxidase by murine embryonic fibroblasts, whereas in vitro studies identify pro-lysyl oxidase as the first known substrate for mTLL-2.     

Inhibition by heparin of the oxidation of lysine in collagen by lysyl oxidase

The generation of covalent cross-linkages in collagen is initiated by the deamination by lysyl oxidase of specific lysine residues in this connective tissue protein. Since lysyl oxidase activity is influenced by ionic ligands bound to its protein substrates, the effect of heparin, an anionic glycosaminoglycan known to bind to collagen, was explored by using collagen and elastin substrates and highly purified lysyl oxidase. Concentrations of heparin up to 1 mg mL-1 had little effect on the enzymatic rate of oxidation if it was added prior to the addition of enzyme to a preformed fibrillar collagen substrate or to an insoluble elastin substrate. However, collagen oxidation was inhibited by 85% if this glycosaminoglycan was present at 0.4 mg mL-1 during collagen fibril formation before addition of the enzyme. Similarly, the rate and extent of collagen fibrillogenesis in the absence of lysyl oxidase were each markedly inhibited in the presence of 0.4 mg mL-1 heparin. Heparin also inhibited the extent of tight binding of lysyl oxidase to preformed fibrils by about 40% under conditions where enzyme activity against preformed fibrils was hardly affected. These results suggest that heparin may modulate the oxidation and thus the insolubilization of extracellular collagen fibers, possibly under conditions where elastin fiber synthesis is not affected, and that the tight binding of lysyl oxidase to collagen is not completely related to the expression of enzyme activity toward this substrate. These results also have mechanistic implications for the retarding effect of heparin on postoperative wound healing.     

A role for lysyl oxidase regulation in the control of normal collagen deposition in differentiating osteoblast cultures

Differentiation of phenotypically normal osteoblast cultures leads to formation of a bone-like extracellular matrix in vitro. Maximum collagen synthesis occurs early in the life of these cultures, whereas insoluble collagen deposition occurs later and is accompanied by a diminished rate of collagen synthesis. The mechanisms that control collagen deposition seem likely to include regulation of extracellular collagen biosynthetic enzymes, but expression patterns of these enzymes in differentiating osteoblasts has received little attention. The present study determined the regulation of lysyl oxidase as a function of differentiation of phenotypically normal murine MC3T3-E1 cells at the level of RNA and protein expression and enzyme activity. In addition, the regulation of BMP-1/mTLD mRNA levels that encodes procollagen C-proteinases was assayed. The role of lysyl oxidase in controlling insoluble collagen accumulation was further investigated in inhibition studies utilizing beta-aminopropionitrile, a specific inhibitor of lysyl oxidase enzyme activity. Results indicate that lysyl oxidase is regulated as a function of differentiation of MC3T3-E1 cells, and that the maximum increase in lysyl oxidase activity precedes the most efficient phase of insoluble collagen accumulation. By contrast BMP-1/mTLD is more constitutively expressed. Inhibition of lysyl oxidase in these cultures increases the accumulation of abnormal collagen fibrils, as determined by solubility studies and by electron microscopy. Taken together, these data support that regulation of lysyl oxidase activity plays a key role in the control of collagen deposition by osteoblast cultures.     

腹腔镜腹股沟疝修补术患者术后出血及二次手术相关因素分析

目的:探讨影响腹腔镜腹股沟疝修补术患者术后出血及二次手术相关因素。方法:回顾性分析行腹腔镜腹股沟疝修补术的6632例患者的临床资料,将凝血病、抗凝治疗或抗血小板治疗的829例患者归入风险组(n=829),其他患者归为对照组(n=5803),收集并比较二组患者的手术方式、年龄、性别、美国麻醉师协会(ASA)分级、疝缺损面积(I-III级)、一期手术与二次手术等患者资料,进行一年随访;采用多变量分析影响患者继发性出血及并发症所致二次手术的相关因素。结果:风险组的术后出血发生率显著高于对照组(4.22%vs 1.26%,P0.001),所有患者术后出血发生率为1.63%。影响术后继发性出血的其它负面因素有:开放式腹股沟疝术式、年龄增加、较高ASA分级、二次手术、男性和较大的疝缺损。风险组与并发症相关的二次手术发生率显著高于对照组(2.65%vs 1.14%,P0.001),所有患者与并发症相关的二次手术发生率为1.32%。影响患者并发症所致二次手术负面因素有:双侧手术、较高ASA分级、凝血病与抗凝治疗和抗血小板治疗、高龄,保护因素包括:较小的疝缺损面积与腹腔镜手术式。结论:行腹腔镜腹股沟疝手术患者术后出血性及并发症相关的二次手术的风险小于行开放术式患者。     

Reduced lysyl oxidase activity in skin fibroblasts from patients with Menkes'' syndrome.

Lysyl oxidase activity against both collagen and elastin substrates has been examined in the culture medium of skin fibroblasts derived from unrelated patients with Menkes' syndrome and from control subjects. The medium of three Menkes' fibroblast lines showed 3--30% of the activity present in the medium of control fibroblasts, against a purified collagen substrate. Lysyl oxidase activity in the culture medium of two of the Menkes' fibroblast lines was also examined by using a crude aortic-elastin substrate and was similarly decreased in comparison with that in the medium of control fibroblasts. Lysyl oxidase activity in the medium of a fourth fibroblast line, derived from a foetus with Menkes' syndrome, was 42% of that in the medium of control fibroblasts derived from a 1-day-old baby against a collagen substrate, and 26% of that in control fibroblast medium against an elastin substrate. The copper content of the cell layers of the Menkes' fibroblast cultures was elevated in comparison with normal fibroblast cultures, as has previously been reported to be characteristic of such cells. It is suggested that the decrease in lysyl oxidase activity would help to explain the connective tissue defects observed in Menkes' syndrome, and that this reduction, in conjunction with the elevated concentrations of cellular copper, would support the hypothesis that a functional intracellular copper deficiency exists in Menkes' syndrome.     

Dietary copper, simple sugars, and metabolic changes in pigs

Inadequate dietary copper is known to result in undesirable metabolic changes in rats and humans. Abnormal cardiac function, leading to sudden death, is a common finding when copper deficient rats are fed a 62% fructose diet. To further study the apparent mineral-carbohydrate relationship to cardiac physiology, 3 male and 3 female swine were randomly assigned to four groups (6 pigs per group) which were fed low copper (1.5 ppm) or copper supplemented (40 ppm) diets with 20% of calories from either fructose or glucose for 10 weeks. In agreement with results from other animal studies, copper deficient swine exhibited decreased plasma ceruloplasmin, erythrocyte superoxide dismutase and plasma lysyl oxidase activities and lowered serum copper. The copper deficient fructose group had the lowest aortic lysyl oxidase activity and hematocrit when compared to the other groups. The relative heart weight in the copper deficient fructose group was 93% greater than the other three dietary groups. The livers of copper deficient fructose fed pigs were also significantly larger. Two enzymes related to cardiac and hepatic function, aspartate and alanine aminotransferase were also measured. Copper deficiency significantly lowered alanine aminotransferase but there was no dietary effect on aspartate amino-transferase. The results of this project indicate that the pig is a sensitive model for the study of cardiovascular abnormalities which occur when fructose is consumed with a low copper diet.     

Effects of dietary vitamin D and calcium on lysyl oxidase activity in chick bone metaphyses.

Activity of lysyl oxidase, an enzyme responsible for production of aldehydic precursors for lysine-derived collagen crosslinks, was measured in tibial metaphyses from chicks receiving different dietary levels of vitamin D and Ca for 2 weeks after hatching. Enzyme activities were increased twofold in D-deficient chicks compared to activities from chicks receiving control levels of vitamin D. Addition of Ca to the D-deficient diet had no effect on lysyl oxidase activity. It is suggested that vitamin D may play a role in the age-related decrease in lysyl oxidase activity that normally occurs in chick bone.     

Regulation of collagen deposition and lysyl oxidase by tumor necrosis factor-alpha in osteoblasts

Tumor necrosis factor-alpha (TNF-alpha) inhibits osteoblast function in vitro by inhibiting collagen deposition. Studies generally support that TNF-alpha does not inhibit collagen biosynthesis by osteoblasts but that collagen deposition is in some way diminished. The study investigated TNF-alpha regulation of biosynthetic enzymes and proteins crucial for posttranslational extracellular collagen maturation in osteoblasts including procollagen C-proteinases, procollagen C-proteinase enhancer, and lysyl oxidase. The working hypothesis is that such regulation could inhibit collagen deposition by osteoblasts. We report that in phenotypically normal MC3T3-E1 osteoblasts, TNF-alpha decreases collagen deposition without decreasing collagen mRNA levels or procollagen protein synthesis. Analyses of the cell layers revealed that TNF-alpha diminished the levels of mature collagen cross-links, pyridinoline and deoxypyridinoline. Further analyses revealed that the mRNA expression for lysyl oxidase, the determining enzyme required for collagen cross-linking, is down-regulated by TNF-alpha in a concentration- and time-dependent manner by up to 50%. The decrease was accompanied by a significant reduction of lysyl oxidase protein levels and enzyme activity. By contrast, Northern and Western blotting studies revealed that procollagen C-proteinases bone morphogenic protein-1 and mammalians Tolloid and procollagen C-proteinase enhancer were expressed in MC3T3-E1 cells and not down-regulated. The data together demonstrate that TNF-alpha does not inhibit collagen synthesis but does inhibit the expression and activity of lysyl oxidase in osteoblasts, thereby contributing to perturbed collagen cross-linking and accumulation. These studies identify a novel mechanism in which proinflammatory cytokine modulation of an extracellular biosynthetic enzyme plays a determining role in the control of collagen accumulation by osteoblasts.     

The long-term effect of mesh bioprosthesis in inguinal hernia repair on testicular nitric oxide metabolism and apoptosis in rat testis

Polypropylene mesh is the most widely used material in inguinal hernia repair. Although polypropylene mesh is known as an inert material, it is experimentally proven that mesh generates a chronic inflammatory tissue reaction. The aim of the present study was to investigate the long-term effects of polypropylene mesh material used in inguinal hernia operations on testicular function, testicular nitric oxide (NO) metabolism and germ cell-specific apoptosis in rats. The study comprised 40 male rats that were randomly allocated into two groups. In group 1, the left spermatic cord was elevated and a 0.5 x 1 cm polypropylene mesh was placed behind the left inguinal spermatic cord and group 2 consisted of the sham-operated controls. Blood samples were taken at 6 months preoperatively and postoperatively after to assess luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels for hormonal evaluation. Testicular NO was evaluated by the Griess method, apoptosis by a TUNEL method and inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) expressions by immunohistochemical staining. Mild (+) eNOS expression was observed in all specimens. Mild (+) iNOS expression was only detected in ipsilateral testis of the mesh-implanted study group. Apoptotic cells were not detected in any samples. We are of the opinion that long-term polypropylene mesh implantation has no effect on testicular hormonal function and only a limited effect on nitric oxide levels and this effect is not sufficient to cause apoptosis in testis that could lead to infertility. It seems that mesh implantation is a reliable method in inguinal hernia repair; however, further work is required by more sensitive methods to fully elucidate the potential testicular damage.     

Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase

The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.