Presence Of Elastin Research Articles

A single-stage bilayered skin reconstruction using Glyaderm® as an acellular dermal regeneration template results in improved scar quality: an intra-individual randomized controlled trial

Absence of almost the entire reticular dermal layer is inherent to the use of autologous split-thickness skin grafting (STSG) to close full-thickness wounds, often resulting in hypertrophic scars and contractures. Many dermal substitutes have been developed, but unfortunately most have varying results in terms of cosmetic and/or functional improvement as well as patient satisfaction, in addition to high costs. Bilayered skin reconstruction using the human-derived glycerolized acellular dermis (Glyaderm®) has been reported to result in significantly improved scar quality using a two-step procedure. Unlike the necessary two-step procedure for most commercially available dermal substitutes, in this study we aimed to investigate the use of Glyaderm® in a more cost-effective single-stage engrafting. This is a method which, if autografts are available, is preferred by the majority of surgeons given the reduction in costs, hospitalization time and infection rate. A prospective, randomized, controlled, intra-individual, single-blinded study was performed, investigating the simultaneous application of Glyaderm® and STSG vs. STSG alone in full-thickness burns or comparable deep skin defects. During the acute phase, bacterial load, graft take and time to wound closure were assessed and were the primary outcomes. Aesthetic and functional results (secondary outcomes) were evaluated at 3, 6, 9 and 12months follow-up using subjective and objective scar measurement tools. Biopsies for histological analysis were taken at 3 and 12months. A total of 66 patients representing 82 wound comparisons were included. Graft take (>95%), pain management and healing time were comparable in both groups. At 1year follow-up, the overall Patient and Observer Scar Assessment Scale assessed by the patient was significantly in favour of sites where Glyaderm® was used. Not infrequently, patients attributed this difference to improved skin sensation. Histological analysis showed the presence of a well-formed neodermis, with donor elastin present for up to 12months. A single-stage bilayered reconstruction with Glyaderm® and STSG results in optimal graft take without loss of Glyaderm® nor the overlaying autografts due to infection. The presence of elastin in the neodermis was demonstrated during long-term follow-up in all but one patient, which is a crucial factor contributing to the significantly improved overall scar quality as evaluated by the blinded patients. The trial was registered on clinicaltrials.gov and received the following registration code: NCT01033604.

Evaluation of Collagen and Elastin Content in Skin of Multiparous Minks Receiving Feed Contaminated with Deoxynivalenol (DON, Vomitoxin) with or without Bentonite Supplementation.

Simple SummaryThe presence of mycotoxins in products intended for consumption is harmful to the health of both people and animals. One of the most abundant mycotoxins in mink’s feed, often contaminating cereal grains, is a mycotoxin produced by the fungi Fusarium spp. deoxynivalenol (DON). The aim of the study was to investigate whether and how the long-term supply of this mycotoxin in feed influences the skin of adult female minks. An additional objective was to assess the effects of the bentonite additive to feed contaminated by DON, which has the ability to reduce the impact of mycotoxins. The scrapes of the skin were collected from animals after euthanasia and before pelting. After preparing histological slides, samples were examined microscopically. The thickness of the epidermis and dermis was investigated and the presence of elastin and collagen. These parameters determine the quality of the fur skins and economic aspect of this animal husbandry. The results showed that DON causes a decrease in the presence of total collagen and absence of immature collagen, thus reducing the elasticity and flexibility of the skin. The addition the bentonite to feed stimulates the production of collagen, restoring the proper relationship between the tested parameters in mink’s skin.Deoxynivalenol (DON, vomitoxin) is considered one of the most dangerous mycotoxins contaminating cereal products for food and feed. One of the protective methods against the adverse effect of DON on mink health is to use a component such as bentonite as a feed supplement to allow toxins absorption. The aim of this study was to determine the effect of DON, administered alone or with bentonite, on the histological structure of the skin and the content of collagen and elastin. A multiparous minks from control group (not exposed to DON) and a study groups receiving fed with DON-containing wheat for seven months: I: at a concentration of 1.1 mg/kg of feed, II: at a concentration of 3.7 mg/kg, III: DON at a concentration of 3.7 mg/kg and bentonite at a concentration of 0.5 kg/1000 kg of feed (0.05%) and IV: DON at a concentration of 3.7 mg/kg and bentonite at a concentration 2 kg/1000 kg (0.2%). After performing euthanasia and before pelting, skin samples of 2 cm in diameter were drawn from the multiparous minks from the lateral surface of the right anterior limb. Our obtained results clearly indicate that DON administered for a period of seven months at a dose of 1.1 mg/kg significantly changes the thickness of skin of a multiparous mink. It causes an increase in the percentage of elastin from 5.9% to 9.4% and a decrease in immature collagen, which results in a change in the collagen/elastin ratio from 10/1 to 5/1. A dose of 3.7 mg/kg DON in feed without or with 0.05% bentonite causes the absence of immature collagen in the dermis, but the addition of 0.2% bentonite in the feed reveals the presence of immature collagen and increase the percentage of the elastin.

Extraction of elastin from tannery wastes: A cleaner technology for tannery waste management

Abstract During leather processing, most of the trimmed raw skins/hides are thrown as wastes which require solid waste management. In the present study, raw hide trimming wastes have been used as the source for extracting elastin. Elastin is extracted from raw trimming wastes through thermo-chemical treatment method using alkali pre-treatments. The physical characterization using differential scanning calorimetry showed the melting point at 104 °C. Elastin has shown higher denaturation temperature above 275 °C and thermal gravimetric shows six mass loss steps. Through thermo-chemical extraction method, 90% of yield has obtained. From various characterization results, it is observed that extracted elastin is chemically confined with its physicochemical properties. Moreover, biocompatibility studies also confirm that the enhancement of cell proliferations in the presence of elastin, which makes it more non-toxic and biocompatible which makes it feasible for tissue engineering applications. The study provides an insight into tapping unused high-value products from tannery wastes.

Electrospun PCL-protein scaffolds coated by pyrrole plasma polymerization

Polymeric scaffolds prepared from polycaprolactone (PCL), PCL-collagen and PCL-elastin were prepared by electrospinning. The scaffolds were coated by plasma polymerization of pyrrole doped with iodine, to improve cellular adhesion and fibroblast proliferation. The morphology, composition, and crystalline structure of the scaffolds were characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy, small- and wide-angle X-ray scattering, thermogravimetric analysis and differential scanning calorimetry. The scaffold fibers had average diameters between 90 and 330 nm before coating. After coating by plasma polymerization, the average diameters increased to between 290 and 530 nm. The presence of elastin and collagen in the fibers was corroborated by infrared spectroscopy. X-ray scattering measurements show that neither elastin nor collagen form crystals in the fiber, while PCL maintains its crystallinity and is not affected by the plasma treatment. Fibroblast cell cultures in the presence of the scaffolds were characterized by viability assays and SEM. The results show that the scaffolds modified with plasma polymerized pyrrole provide a suitable environment for fibroblast adhesion, growth and viability.

Cellular response to collagen-elastin composite materials

Collagen is used extensively in tissue engineering due to its biocompatibility, near-universal tissue distribution, low cost and purity. However, native tissues are composites that include diverse extracellular matrix components, which influence strongly their mechanical and biological properties. Here, we provide important new findings on the differential regulation, by collagen and elastin, of the bio-response to the composite material. Soluble and insoluble elastin had differing effects on the stiffness and failure strength of the composite films. We established that Rugli cells bind elastin via EDTA-sensitive receptors, whilst HT1080 cells do not. These cells allowed us to probe the contribution of collagen alone (HT1080) and collagen plus elastin (Rugli) to the cellular response. In the presence of elastin, Rugli cell attachment, spreading and proliferation increased, presumably through elastin-binding receptors. By comparison, the attachment and spreading of HT1080 cells was modified by elastin inclusion, but without affecting their proliferation, indicating indirect modulation by elastin of the response of cells to collagen. These new insights highlight that access to elastin dominates the cellular response when elastin-binding receptors are present. In the absence of these receptors, modification of the collagen component and/or physical properties dictate the cellular response. Therefore, we can attribute the contribution of each constituent on the ultimate bioactivity of heterogeneous collagen-composite materials, permitting informed, systematic biomaterials design. Statement of SignificanceIn recent years there has been a desire to replicate the complex extracellular matrix composition of tissues more closely, necessitating the need for composite protein-based materials. In this case both the physical and biochemical properties are altered with the addition of each component, with potential consequences on the cell. To date, the different contributions of each component have not been deconvolved, and instead the cell response to the scaffold as a whole has been observed. Instead, here, we have used specific cell lines, that are sensitive to specific components of an elastin-collagen composite, to resolve the bio-activity of each protein. This has shown that elastin-induced alteration of the collagen component can modulate early stage cell behaviour. By comparison the elastin component directly alters the cell response over the short and long term, but only where appropriate receptors are present on the cell. Due to the widespread use of collagen and elastin, we feel that this data permits, for the first time, the ability to systematically design collagen-composite materials to promote desired cell behaviour with associated advantages for biomaterials fabrication.

Nanofibrous Scaffolds with Biomimetic Composition for Skin Regeneration

Treatments of skin injuries caused by trauma and diseases are among the most considerable medical problems. The use of scaffolds that can cover the wound area and support cellular ingrowth has shown great promise. However, mimicking the physicochemical properties of the native skin extracellular matrix (ECM) is essential for the successful integration of these scaffolds. Elastin has been known as the second main protein-based component of the native skin ECM. In this research, scaffolds containing gelatin, cellulose acetate, and elastin were fabricated using electrospinning. Subsequently, the effects of soluble elastin on the physical, mechanical, and biological properties of the prepared scaffolds were studied. The results confirmed that the presence of elastin in the composition changed the fiber morphology from straight to ribbon-like structure and decreased the swelling ratio and degradation rate of the scaffold. In vitro experiments showed that elastin-containing scaffolds supported the attachment and proliferation of fibroblast cells. Overall, the obtained results suggest the ternary blend of gelatin, cellulose acetate, and elastin as a good candidate for skin tissue engineering.

Human pulmonary artery endothelial cells in the model of mucopolysaccharidosis VI present a prohypertensive phenotype

BackgroundMucopolysaccharidosis type VI (MPS VI) is an autosomal recessive lysosomal disorder caused by a deficient activity of N-acetylgalactosamine-4-sulfatase (ARSB). Pulmonary hypertension (PH) occurs in MPS VI patients and is a marker of bad prognosis. Malfunction of endothelium, which regulates vascular tonus and stimulates angiogenesis, can contribute to the occurrence of PH in MPS VI. AimThe aim of the study was to establish a human MPS VI cellular model of pulmonary artery endothelial cells (HPAECs) and evaluate how it affects factors that may trigger PH such as proliferation, apoptosis, expression of endothelial nitric oxide synthase (eNOS), natriuretic peptide type C (NPPC), and vascular endothelial growth factor A (VEGFA). ResultsIncreasing concentrations of dermatan sulfate (DS) reduce the viability of the cells in both ARSB deficiency and controls, but hardly influence apoptosis. The expression of eNOS in HPAECs is reduced up to two thirds in the presence of DS. NPPC shows a biphasic expression reaction with an increase at 50μg/mL DS and reduction at 0 and 100μg/mL DS. The expression of VEGFA decreases with increasing DS concentrations and absence of elastin, and increases with increasing DS in the presence of elastin. ConclusionOur data suggest that MPS VI endothelium presents a prohypertensive phenotype due to the reduction of endothelium's proliferation ability and expression of vasorelaxing factors.

Hepatic Fibrosis in a Long-term Murine Model of Sepsis

Chronic sequelae of sepsis represent a major, yet underappreciated clinical problem, contributing to long-term mortality and quality-of-life impairment. In chronic liver disease, inflammation perpetuates fibrogenesis, but development of fibrosis in the post-acute phase of systemic inflammation has not been studied. Therefore, a mouse model of post-acute sequelae of sepsis was established based on polymicrobial peritonitis under antibiotic protection. Survival decreased to approximately 40% within 7 days and remained constant until day 28 (post-acute phase). In survivors, clinical recovery was observed within 1 week, whereas white blood cell and platelet count, as well as markers of liver injury, remained elevated until day 28. Macroscopically, inflammation and abscess formation were detected in the peritoneal space and on/in the liver. Microscopically, acute-chronic inflammation with ductular proliferation, focal granuloma formation in the parenchyma, and substantial hepatic fibrosis were observed. Increased numbers of potentially pathogenetic macrophages and α-smooth muscle actin-positive cells, presumably activated hepatic stellate cells, were detected in the vicinity of fibrotic areas. Fibrosis was associated with the presence of elastin and an augmented production/deposition of collagen types I and III. Microarray analyses revealed early activation of canonical and noncanonical pathways of hepatic stellate cell transdifferentiation. Thus, chronic sequelae of experimental sepsis were characterized by abscess formation, persistent inflammation, and substantial liver injury and fibrosis, the latter associated with increased numbers of macrophages/α-smooth muscle actin-positive cells and deposition of collagen types I and III. This suggests persistent activation of stellate cells, with consecutive fibrosis-a hallmark of chronic liver disease-as a result of acute life-threatening infection.

Tissue Augmentation with Fibrin Sealant and Cultured Fibroblasts: A Preliminary Study

Nonoperative subdermal tissue augmentation is one of the most frequently performed procedures in plastic surgery and dermatological practice. Many products, from biological to synthetic filler substances, are currently available. However, none has achieved ideal clinical efficacy, especially regarding volume maintenance and longevity. We examined the use of fibrin sealant as a biological and fully degradable matrix for dermal augmentation in combination with precultured human fibroblasts and hyaluronic acid gel (HYAFF). Four implant preparations were studied: fibrin glue only (F); 1% HYAFF mixed in fibrin glue (FH); 1.8×10(6)cells/ml of fibrin glue (FC); and 1% HYAFF and 1.8×10(6)cells/ml of fibrin glue (FHC). Each mouse was given two separate subcutaneous injections of implant material. At 1, 3, and 6weeks two mice from each group were sacrificed, such that there was an n=4 for each implant group at each time point. The mice were grossly examined for implant retention and the implants were evaluated by means of immunohistochemistry for fibrosis, integration into surrounding tissue, presence of elastin, and blood vessel infiltration. Only the implants in the cell-containing groups, FC and FHC, remained after 6weeks. Moreover, with the exception of a mild inflammatory response, no adverse affects of the cell-seeded implants were noted. Presence of fibroblasts increases implant durability. Further studies should evaluate the ideal hyaluronic acid and fibroblast concentration for long-term longevity.

The effect of elastin on chondrocyte adhesion and proliferation on poly (ɛ-caprolactone)/elastin composites

The aim of this study was to demonstrate the effect of elastin on chondrocyte adhesion and proliferation within the structure of poly (ɛ-caprolactone) (PCL)/elastin composites. The homogenous 3D structure composites were constructed by using high pressure CO 2 in two stages. Porous PCL structures with average pore sizes of 540 ± 21 μm and a high degree of interconnectivity were produced using gas foaming/salt leaching. The PCL scaffolds were then impregnated with elastin and cross-linked with glutaraldehyde (GA) under high pressure CO 2. The effects of elastin and cross-linker concentrations on the characteristics of composites were investigated. Increasing the elastin concentration from 25 mg/ml to 100 mg/ml elevated the amount of cross-linked elastin inside the macropores of PCL. Fourier transform infrared (FTIR) analysis showed that elastin was homogeneously distributed throughout the 3D structure of all composites. The weight gain of composites increased 2-fold from 15.8 ± 0.3 to 38.3 ± 0.7 (w/w) % by increasing the elastin concentration from 25 mg/ml to 50 mg/ml and approached a plateau above this concentration. The presence of elastin within the pores of PCL improved the water uptake properties of PCL scaffolds; the water uptake ratio of PCL was enhanced 100-fold from 0.030 ± 0.005 g liquid/g polymer to 11.80 ± 0.01 g liquid/g polymer, when the elastin solution concentration was 50 mg/ml. These composites exhibited lower compressive modulus and energy loss compared to pure PCL scaffolds due to their higher water content and elasticity. In vitro studies show that these composites can support primary articular cartilage chondrocyte adhesion and proliferation within the 3D structures. These results demonstrate the potential of using PCL/elastin composites for cartilage repair.

Use of a proteomics approach to identify favourable conditions for production of good quality lambskin leather

It is necessary to understand the changes that occur during the initial processing of lamb skins, because these will affect the final quality of the leather. The types of collagen, their macro and micro structures, the presence of proteins other than collagens, and the quantity and the type of proteoglycans, all have a profound effect on the quality of leather. Proteins isolated from untreated or raw sheep skin and from pickled skin (skins treated with sodium sulfide and lime followed by bating with enzymes, then preserved in sodium chloride and sulfuric acid) were significantly different when analysed by use of 2D gel electrophoresis and mass spectrometry. Agarose gel electrophoresis with a very sensitive sequential staining procedure has been used to identify the glycosaminoglycans present in raw and treated skin and their impact on quality of leather. Results showed that effective removal of proteoglycans acting as inter-fibrillar adhesives of collagen fibrils seemed to improve leather quality. Removal of these molecules not only opens up the fibre structure of the skin but may also be important in wool removal. The presence of elastin, which imparts elastic properties to skin, is of significant importance to tanners. The amino acids desmosine and isodesmosine, found exclusively in elastin, were quantitatively analysed to assess the role of elastin in leather quality.

Quantification of Elastic Recoil after Balloon Angioplasty in the Iliac Arteries

Elastic recoil of the arterial wall has been shown to be responsible for a significant loss of luminal area after balloon angioplasty in the coronary arteries, but it has not been well studied in the peripheral arteries. Because elastic recoil depends on the presence of elastin in the arterial wall, and the amount of elastin varies by artery and proximity to the aorta, the importance of this response to angioplasty may be different in peripheral arteries. The purpose of this study is to document the degree of elastic recoil in the iliac arteries, and analyze variables that might influence the results. A series of 19 patients with 25 iliac artery stenoses underwent balloon angioplasty followed by placement of a Palmaz stent with the same-sized angioplasty balloon. The minimum luminal diameter of the lesion was measured before treatment, immediately after balloon angioplasty, and again after stent placement. The arterial diameter after stent placement was defined as the diameter of the inflated balloon. The degree of recoil was correlated with nine variables: patient age and sex, lesion location and length, lesion severity (as percent stenosis), the balloon:artery ratio, and three factors related to lesion morphology--complex versus simple, eccentric versus concentric, and calcified versus noncalcified. Elastic recoil averaged 36% +/- 11% and ranged from 19% to 54% in this series of patients. The only variable that significantly influenced the degree of elastic recoil was the balloon:artery ratio (P =.039), which was directly related. Elastic recoil is a significant limitation of balloon angioplasty in the iliac arteries. This study illustrates the importance of techniques that limit recoil, such as vascular stents, in angioplasty of the iliac arteries.

High glutaraldehyde concentrations mitigate bioprosthetic root calcification in the sheep model

Background. Fixation at high glutaraldehyde (GA) concentrations mitigated bioprosthetic calcification in the rat model. The present study intended to verify this observation in the circulatory sheep model. Methods. Porcine aortic roots were either fixed in 0.2%, 1.0%, or 3.0% GA. Eight roots per group were implanted in the distal aortic arch of sheep. After six weeks and six months calcification and inflammation were quantitatively and qualitatively assessed. Results. By increasing the GA concentration from 0.2% to 3.0%, aortic wall calcification could be reduced by 38% after 6 weeks and 34% after 6 months of implantation ( p < 0.01). Mineralization coincided with the presence of elastin although calcium was predominantly found in cell nuclei and membranes. Leaflet calcification was absent in all groups after 6 weeks but in a few leaflets presented as heterogeneous, nodular spongiosa deposits after 6 months. Overall, differences between 0.2%-, 1.0%-, and 3.0%-fixed tissue were quantitative but not qualitative regarding distribution patterns. There was no significant difference in inflammatory host reaction between all groups. Conclusions. We have shown in the circulatory sheep model that the anticalcific effect of better cross-linking seems to outweigh the intrinsic pro-calcific effect of GA accumulation in bioprosthetic aortic wall tissue.

Effects of porcine pancreatic elastase-1 on elastin in human trabecular meshwork

Human trabecular meshwork tissue was embedded in Lowicryl K4M and subjected to electron microscopy. Elastin in the trabecular meshwork was stained using protein A-gold immunostaining method and observed under an electron microscope. For the enzymatic digestion study, the tissue specimens were incubated in various concentrations of solutions of both porcine pancreatic elastase-1 (PPE) and {ie297-1} complex ({ie297-2}), before immunostaining. Many gold particles showing the presence of elastin were found within elastic fibers of the trabecular meshwork. The number of gold particles in the elastic fibers significantly decreased after the enzymatic digestion.

Production of serine chymotrypsin-like elastase by Aspergillus fumigatus strains

Thirty-four Aspergillus fumigatus strains isolated from air, horse-hair; agricultural soil and human samples were screened to evaluate the production of elastase. Aspergillus fumigatus strains were grown in elastin solid medium, showing a widespread elastin solubilization. However, isolates from human and agricultural soil samples were found to be the highest elastase producers. Then, eight out of 34 strains were grown in four different liquid media, on wich we investigated total and specific proteolytic activity. Results from this experiments suggest that the elastase production is induced by the presence of elastin as a substrate and that the elastase is a chymotrypsin like enzyme. Inhibitory profile showed that the A.fumigatus elastase is a serine proteinase.

Human leucocyte migration through collagen matrices containing other extracellular matrix components

The purpose of this study was to investigate the migration of human peripheral blood leucocytes through collagen gels containing extracellular matrix components in vitro. This further extends the findings of a previous study ( Reid et al, 1990 ), using rabbit peritoneal neutrophils, in which it was found that increasing the mechanical strength of the gels decreased the cell migration. In the current study it was found that human neutrophil and lymphocyte migration through collagen matrices was inhibited by increasing the collagen concentration, whereas the presence of serum had no effect on migration. Incorporation of chondroitin sulphate into collagen gel matrices resulted in a slight increase in leucocyte migration. The presence of elastin had no effect on the migration of leucocytes through collagen matrices, whereas incorporation of hyaluronate into collagen matrices decreased leucocyte migration. The morphology of these matrices suggests that altered migration was not due to differences in pore size between fibres.

Kinetics of the inhibition of free and elastin-bound human pancreatic elastase by α1-proteinase inhibitor and α2-macroglobulin

At pH 8.0 and 25°C α 1-proteinase inhibitor and α 2-macroglobulin bind human pancreatic elastase with rate constants of 4.7·10 5 M −1·s −1 and 6.4·10 6 M −1·s −1, respectively. The corresponding delay times of elastase inhibition in plasma are 0.4 s and 0.2 s, respectively, indicating that both inhibitors may act as physiological antielastases. Elastin impairs the elastase inhibitory capacity of α 1-proteinase inhibitor and α 2-macroglobulin. In presence of human elastin, the former behaves like a slow-binding elastase inhibitor, with a rate constant of about 260 M −1·s −1. In contrast, α 2-macroglobulin is a fast-binding inhibitor of elastin-bound elastase, but only one of its two sites is functioning in presence of elastin.

Association of elastin with pseudoexfoliative material: an immunoelectron microscopic study.

Using immunoelectron microscopy, the presence of elastin and tropoelastin was demonstrated in pseudoexfoliative (PSX) material in all its classical sites on the lens capsule, ciliary non-pigment epithelium, iris epithelium and stroma, and conjunctiva. Some variability in binding affinity was seen in different sites, and labelling was more often on the periphery than the center of the PSX fibers. The elastin epitope on PSX material was more sensitive to processing than the remarkably stable epitope on mature elastic fibers. Since neither elastin nor a related component of PSX fibers, elastic microfibrillar protein, is a circulating protein, both are likely to be secreted by local ocular cells. Most of these local cells are not involved in elastogenesis normally, suggesting that an abnormal stimulus or defective regulation of matrix synthesis exists in this disease.

Sputum Elastin Fibers and the Diagnosis of Necrotizing Pneumonia

Potassium hydroxide (KOH) preparations for elastin fibers on sputum obtained from 80 patients seen over a four-month period at two Cleveland hospitals were performed. The results were compared with roentgenographic evidence of necrosis and case diagnosis. Sixty-one patients had neither elastin in sputum nor roentgenographic evidence of cavitation; 11 had positive results using both methods. Two patients had no elastin fibers in sputum but had parenchymal pulmonary cavities on chest x-ray film. Six patients had elastin observed in KOH preparations of sputum, but no cavitation roentgenographically. The presence of elastin in sputum was strongly correlated with roentgenographic evidence of pulmonary necrosis (p = 5.7 X 10(-8]. Including patients seen before, after, and during the prospective study, we have observed a total of nine with positive sputum preparations for elastin and no cavitation on chest x-ray film for whom tissue was available for study. All had pulmonary necrosis histologically. Our observations suggest that the KOH preparation of sputum for elastin fibers may be more sensitive than the chest roentgenogram in the detection of pulmonary necrosis and may be a useful adjunct in the diagnosis of necrotizing disease.

Strongyloides ratti and Trichinella spiralis: Net charge of epicuticle

The intact epicuticles of Strongyloides ratti stage-3 larvae and Trichinella spiralis stage-1 larvae were found to have a surface net negative charge. Ultrastructural studies on S. ratti using cationized ferritin and ruthenium red showed the negative charge to be dense and uniformly distributed over the epicuticular surface. Staining with acetic acid-ferric oxide hydrosol occurred at pH 1.65 and suggests that amino acid carboxyl groups were not responsible for the negative charge property. Alcian blue staining occurrred at pH 0.5 and at a critical electrolyte concentration (CEC) of 0.9 M MgCl 2, a property similar to that of highly sulfated mucopolysaccharides such as the proteoglycan keratan sulfate. In contrast, T. spiralis larvae failed to stain with alcian blue below pH 5.0 or at a CEC of 0.1 M, suggesting its negative charge is associated with dissociated amino acid carboxyl groups. Attempts to remove the negative charge-bearing components in the epicuticle of S. ratti by detergents, organic solvents, denaturing agents, proteases, uronidases, neuraminidases, and lipases were unsuccessful. The presence of elastin in the S. ratti larval outer cortical layer was indicated by its vulnerability to elastase and its reaction to aldehyde fuchsin-alcian blue stain. These results show that the epicuticle of S. ratti is not a typical cell membrane, although it appears to have ultrastructural similarities. It is suggested that the association of highly sulfated mucopolysaccharides with the epicuticular surface of free-living nematodes such as S. ratti L 3 may reflect a greater need to protect against surface desiccation. It is also postulated that the highly negatively charged surface may have anticomplementary and anticoagulation effects.