Collagen Pattern Research Articles

Effects of olmesartan and enalapril at low or high doses on cardiac, renal and vascular interstitial matrix in spontaneously hypertensive rats

We have evaluated the effects of different doses of an angiotensin‐converting enzyme (ACE) inhibitor, enalapril (ENA) and of an angiotensin II type 1 receptor blocker olmesartan (OLM), on extracellular matrix of the heart, kidney, aorta and mesenteric artery of spontaneously hypertensive rats (SHR). Forty SHR and eight Wistar–Kyoto controls (WKY) were included in the study. Eight SHR were treated with high‐dose OLM 15 mg/kg per day, eight with high‐dose ENA 25 mg/kg per day, eight with low‐dose OLM 1 mg/kg per day and eight with low‐dose ENA (2 mg/kg per day). Eight SHR and eight WKY were kept untreated as controls. Treatment was from age 4 to 12 weeks. Systolic blood pressure (SBP) was measured non‐invasively every week. The left ventricular weight to body weight (RLVM) was measured, and the cardiac, aortic and glomerular interstitial collagen content was evaluated using Sirius red staining and image analysis. Mesenteric small arteries were dissected and mounted on a micromyograph, and the media:lumen ratio (M/L) was calculated. Collagen subtypes were evaluated by polarized light microscopy. The SHR treated with high‐dose OLM or ENA showed a normalization of SBP. The RLVM was significantly increased in untreated SHR compared with untreated WKY, whereas significantly lower values were observed in the groups of SHR treated with high‐dose OLM or ENA. A significant increase in cardiac and glomerular collagen content was observed in untreated SHR. Both high‐ or low‐dose OLM and ENA normalized collagen content in the heart and the kidney. Both high‐dose OLM and high‐dose ENA normalized M/L ratio; however, OLM proved to be more effective than ENA in normalizing collagen pattern. In fact, aortic collagen content was normalized by both high‐dose and low‐dose OLM, but only by high‐dose ENA. In conclusion, both OLM and ENA were significantly and equally effective in the prevention of cardiac and renal damage in SHR, whereas OLM was more effective than ENA in terms of effects on vascular extracellular matrix.

Lipoic acid prevents collagen abnormalities in tail tendon of high‐fructose‐fed rats

Non-enzymatic glycation of proteins, leading to chemical modification and cross-linking are of importance in the pathology of diabetic complications. We studied the effect of alpha-lipoic acid (LA) on the glycation and cross-linking of collagen from tail tendon of high-fructose-fed rats. The rats were divided into four groups of six each. Two groups of rats were fed with a high-fructose diet (60/100 g diet) and administered with either LA (35 mg/kg body weight, intraperitoneally) (FRU + LA) or saline (FRU) for 45 days. The other two groups were fed control diet containing starch (60/100 g diet) and administered with either saline (CON) or LA (CON + LA). The rats were maintained for 45 days and then sacrificed. Collagen was isolated from tail tendon and the extent of glycation, fluorescence, aldehyde and peroxidation levels were measured. The tail tendons were separated, and shrinkage temperature was also measured. Acid-soluble collagen was extracted from tail tendon and subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis. Fructose administration caused accumulation of collagen in tail tendon. Glycation, collagen-linked fluorescence, aldehyde and peroxide contents were elevated. The gel pattern of acid-soluble collagen from the fructose-fed rat showed elevated beta-component. These changes were alleviated by the simultaneous administration of LA. Administration of LA has a positive influence on tail tendon collagen glycation and other variables in high-fructose-fed rats.

Expression patterns of collagen I (α1) encoding gene and muscle‐specific genes reveal that the lateral domain of the fish somite forms a connective tissue surrounding the myotome

Somites are repeated, epithelial structures that are derived from the unsegmented paraxial mesoderm located lateral to the notochord. In higher vertebrates, somites differentiate into a sclerotome that subsequently forms the vertebrae and the ribs and into a dermomyotome that gives rise to a myotome, from which arises the skeletal muscle, and to a dermatome, from which arises the dermis. Fish somites have been shown to produce a sclerotome and a myotome, but very little is known regarding their participation in the formation of connective tissues, especially at the junction between the epidermis and the myotome. To investigate the formation of connective tissues in fish somites, we have examined the expression pattern of the collagen I (alpha1) chain. As somitogenesis proceeds rostrocaudally, collagen I (alpha1) expression marks the sclerotomal cells and delineates the formation of the vertebrae. Surprisingly, after the completion of the segmentation, transcript for the collagen I (alpha1) chain appeared in a distinct epithelial-like monolayer situated at the periphery of the developing somite facing the surface epidermis. This epithelial monolayer of somitic cells that covered the superficial slow muscle cells, did not express the myogenic transcriptional regulator myogenin and was devoid of contractile filament. As the somite increased in size, these collagen-expressing epithelial cells flattened, forming a thin cellular layer underlying the epidermis and recovering the lateral surface of the myotome. In conclusion, the lateral domain of the fish somite forms a distinct epithelial cell layer sharing many characteristics with amniote dermatome.

In vitro analysis of matrix proteins and growth factors in dedifferentiating human chondrocytes for tissue-engineered cartilage

ConclusionsWith ongoing culture and dedifferentiation of chondrocytes, significant changes in the expression patterns of various collagens and the insulin-like growth factor (IGF) receptor were detected. The latter could play an important role in the differentiation of human chondrocytes.ObjectiveTissue engineering represents a promising method for the construction of autologous chondrogenic grafts for reconstructive surgery. So far, little is known about the expression of markers for cell proliferation and differentiation in cultured chondrocytes.Material and methodsHuman chondrocytes were isolated from septal cartilage (n=5) and held in primary cell culture. Cells were harvested after 24 h and 6 days. Proliferation was analyzed using an Alamar Blue assay. The differentiation of the cells was investigated using bright field microscopy, the expression patterns of various proteins using immunohistochemistry and the expression of distinct genes using a microarray technique.ResultsThe chondrocytes showed strong proliferation (Day 0: 16.7±0.7 fluorescent units; Day 5: 52.4±2.2 fluorescent units) from the third day of cell culture in medium without growth factors. From this point onwards, a dedifferentiation of the chondrocytes could be observed. In cell culture, the chondrocytes expressed collagen 1 and 10 without expression of collagen 3. After 6 days of cell culture, they expressed collagen 2. The chondrocytes showed constant low expression of the fibroblast growth factor-2 receptor, but constant high expression of vascular endothelial growth factor, matrix metalloproteinase (MMP)2 and MMP9. The cells never expressed the epidermal growth factor receptor. The proportion of IGF receptor-expressing cells diminished significantly during cell culture.

Localization of Type IV Collagen Alpha Chains in the Basement Membrane of Ameloblastoma, Tooth Germ and Oral Mucosa by Using Indirect Immunofluorescence.

Introduction Type IV collagen is a major structural component of basement membrane (BM) and acts as a scaffold for other BM constituents. It is a heterotrimeric molecule that exists in six genetically distinct forms, α1(IV) α6(IV). The ameloblastoma is the most frequently encountered odontogenic epithelial tumor. The BM zone of the ameloblastoma remains a subject of research interest primarily because of increasing evidence of its mediatory role during oncogenesis. However, the expression pattern of specific collagen α (IV) chains in the ameloblastoma BM has not been previously reported. In this preliminary study, indirect immunofluorescence was utilized to localize α1(IV) α6(IV) chains in the BM of two

P56 Relationship between serum biomarkers of type II collagen (C2C; C1,2C and CP II) and radiological patterns in patients with hip osteoarthritis

P56 Relationship between serum biomarkers of type II collagen (C2C; C1,2C and CP II) and radiological patterns in patients with hip osteoarthritis

Expression pattern of collagen XVIII and its cleavage product, the angiogenesis inhibitor endostatin, at the fetal–maternal interface

Endostatin, the C-terminal fragment of the basement membrane protein collagen XVIII regulates epithelial cell migration and impairs tumour growth by inhibiting angiogenesis. Here, we investigated the expression pattern of collagen XVIII/endostatin in human placental and decidual tissues of various ages of gestation as well as in primary villous cytotrophoblasts, trophoblast cell lines, and villous explant cultures differentiating along the invasive pathway. RT-PCR analysis revealed production of collagen XVIII mRNA in total placenta and decidua of early and late pregnancy and in SGHPL-5 and HTR-8/Svneo cells. Collagen XVIII transcripts were absent from purified extravillous trophoblasts and syncytialising trophoblast cultures. Accordingly, an antibody against a protein domain common to different collagen XVIII isoforms detected the 180 kDa protein in villous and decidual tissue and cultivated placental fibroblasts but not in the different isolated trophoblast cell types. Immunohistochemical analyses localised collagen XVIII to villous basement membranes and to the endothelium as well as to placental and decidual stromal cells. Interestingly, expression of various forms of endostatin (20 and 26 kDa) was detected in placenta and decidua using Western blot analyses. Moreover, supplementation of recombinant endostatin increased MMP-2 expression in villous explant cultures and SGHPL-5 cells suggesting that the inhibitor may modulate extravillous trophoblast differentiation.

Characterisation of acid-soluble collagen from skin and bone of bigeye snapper ( Priacanthus tayenus)

The compositions and some properties of acid-soluble collagens (ASC) of the skin and bone of bigeye snapper, ( Priacanthus tayenus) were investigated. The collagens were extracted with the yields of 10.94% and 1.59% on the basis of wet weight, from skin and bone, respectively. Similar electrophoretic patterns of collagens from the skin and bone were observed. Both collagens comprised two different α chains, α1 and α2 and were classified as type I collagen. However, peptide maps of collagen from the skin and bone of bigeye snapper, digested by V8 protease and lysyl endopeptidase, revealed differences between collagens from skin and bone, and both were completely different from those of calf skin collagen. Collagen rehydrated in acetic acid had lower T max and enthalpy than those rehydrated in deionised water, suggesting a conformational change caused by acid. Collagens form the skin and bone had the highest solubility at pH 2 and 5, respectively. No changes in solubility were observed in the presence of NaCl up to 3% (w/v). However, a sharp decrease in solubility was found with NaCl above 3% (w/v).

Fibrillin and other matrix proteins in mitral valve prolapse syndrome

Background Unlike myxomatous degeneration in Marfan syndrome, which has been reported to result from a mutation in the gene that codes for the extracellular structural protein fibrillin, no specific molecular abnormality has been documented to be the underlying cause of myxomatous degeneration in mitral valve prolapse syndrome (MVPS). The present study examined the distribution of fibrillin and other extracellular matrix proteins in patients with isolated MVPS. Methods Mitral valve leaflets from 7 MVPS patients and 5 rheumatic heart disease (RHD) patients were characterized immunohistochemically for fibrillin, elastin, collagen I, and collagen III distribution, and compared with five normal mitral valves. Results In normal mitral valve leaflets immunostaining for fibrillin, elastin, collagen I, and collagen III revealed a fibrillary and laminar pattern in the atrialis and the spongiosa. In addition, both the collagens were present in the ventricularis, and the coarse bundles in the fibrosa exhibited alternating bandlike collagen I immunoreactivity. The staining patterns of fibrillin, elastin, and collagens I and III revealed distinctly different distribution in MVPS relative to the normal and RHD leaflets. MVPS leaflets in areas of myxoid degeneration displayed a more diffuse, weaker, and nonlaminar pattern of staining for fibrillin. Similar, but less severe abnormality of elastin, collagen I, and collagen III was also observed. Unlike diffuse abnormality in MVPS, the disruption of extracellular proteins in RHD only occurred at the site of the inflammatory damage, but the overall architecture was preserved. Conclusions The results of the current study suggest a primary role for abnormal fibrillin and other matrix proteins in producing myxoid degeneration of mitral valve leaflets in MVPS.

Retracted] Fructose Diet‐Induced Skin Collagen Abnormalities Are Prevented by Lipoic Acid

Nonenzymatic glycation of proteins, leading to chemical modification and cross-linking are of importance in the pathology of diabetic complications.We studied the effect of α-lipoic acid (LA) on the content and characteristics of the protein collagen from skin of high-fructose fed rats. The rats were divided into 4 groups of 6 each. Two groups of rats were fed with a high fructose diet (60 g/100 g diet) and administered either LA (35 mg/kg b.w., i.p) (FRU+LA) or 0.2 ml vehicle (saline) (FRU) for 45 days. The other 2 groups were fed with control diet containing starch (60 g/100 g diet) and administered either saline (CON) or lipoic acid (CON+LA). The rats were maintained for 45 days and then sacrificed. Plasma glucose, insulin, fructosamine, protein glycation, and blood glycated hemoglobin (HbA1C) were measured. Collagen was isolated from skin and the physicochemical properties of collagen were studied. Fructose administration caused accumulation of collagen in skin. Extensive cross-linking was evidenced by enhanced glycation and AGE-linked fluorescence. Increased peroxidation and changes in physicochemical properties such as shrinkage temperature, aldehyde content, solubililty pattern, susceptibility to denaturing agents were observed in fructose-fed rats. SDS gel pattern of collagen from these rats showed elevated β component of type I collagen. These changes were alleviated by the simultaneous administration of LA. Administration of LA to fructose-fed rats had a positive influence on both quantitative and qualitative properties of collagen. The results suggest a mechanism for the ability of LA to delay diabetic complications.

The molecular basis of classic Ehlers-Danlos syndrome: A comprehensive study of biochemical and molecular findings in 48 unrelated patients

Classic Ehlers-Danlos syndrome (EDS) is characterized by fragile and hyperextensible skin, atrophic scarring, and joint hypermobility. Mutations in the COL5A1 and the COL5A2 gene encoding the alpha1(V) and the alpha2(V) chains, respectively, of type V collagen have been shown to cause the disorder, but it is unknown what proportion of classic EDS patients carries a mutation in these genes. We studied fibroblast cultures from 48 patients with classic EDS by SDS-PAGE for the presence of type V collagen defects. An abnormal collagen pattern was detected in only 2 out of 48 cell lines, making this a poor method for routine diagnostic evaluation. A total of 42 out of 48 (88%) patients were heterozygous for an expressed polymorphic variant in COL5A1. cDNA from 18 (43%) of them expressed only one COL5A1 allele. In 37 patients, the COL5A1/A2 genes were then analyzed by SSCP and conformation sensitive gel electrophoresis (CSGE). A total of 26 patients that were mutation-negative after SSCP/CSGE screening were reanalyzed by dHPLC. In addition, 11 other patients were analyzed by dHPLC only. In total, 17 mutations leading to a premature stop codon and five structural mutations were identified in the COL5A1 and the COL5A2 genes. In three patients with a positive COL5A1 null-allele test, no causal mutation was found. Overall, in 25 out of 48 patients (52%) with classic EDS, an abnormality in type V collagen was confirmed. Variability in severity of the phenotype was observed, but no significant genotype-phenotype correlations emerged. The relatively low mutation detection rate suggests that other genes are involved in classic EDS. We excluded the COL1A1, COL1A2, and DCN gene as major candidate genes for classic EDS, since no causal mutation in these genes was found in a number of patients who tested negative for COL5A1 and COL5A2.

Increased growth factor production in a human prostatic stromal cell culture model caused by hypoxia.

Local hypoxia may be one of the triggers of embryonic reawakening of the stroma and subsequent hyperplastic growth in the prostate. Using a cell culture model of human prostatic stromal cells, we investigated the effects of hypoxia on activation of hypoxia-inducible factor 1 (HIF 1) and on the production of growth factors. Primary prostatic stromal cells were grown in normal and hypoxic (1% O(2)) atmosphere. Activation of HIF 1 was evaluated after different time intervals by Western blot. Induced secretion of growth factors VEGF, FGF-7, TGF-beta, IL 8, and FGF-2 were analyzed by ELISA. To confirm the in vitro findings we also performed immunohistochemistry of HIF 1alpha as well as pro-collagen I, collagens I, III, and IV in the benign tissue of radical prostatectomy specimens. HIF 1 is activated in a time-dependent manner, already starting 1 hr after exposure of stromal cells to hypoxic conditions. Secretion of VEGF, FGF-7, TGF-beta, FGF-2, and IL 8 is increased under hypoxic in vitro conditions in comparison to normoxia. Levels of TGF-beta, VEGF, and IL 8 were rapidly and statistically significantly increased in the supernatant of hypoxic cells. Consistent with the in vitro findings, immunohistochemistry of HIF 1alpha in (benign prostatic hyperplasia) BPH tissue revealed strong HIF 1alpha nuclear staining in hyperplastic areas. No difference was observed in the collagen pattern between hyperplastic and normal prostate tissue. Prostatic stromal cells respond to hypoxia by upregulation of secretion of several growth factors suggesting that hypoxia can trigger prostatic growth. Therefore, hypoxia might be a key factor contributing to the pathogenesis of BPH.

Expression patterns of cartilage collagens and Sox9 during mouse heart development

A majority of congenital heart defects are due to abnormal development of the valves and membranous septa, i.e., connective tissue components of the heart. During development, an interesting feature of cardiac connective tissue is transient expression of collagens typical for cartilage. To better understand the role of these collagens in the heart, we have performed a systematic study on the temporospatial expression of type II and IX collagen isoforms during mouse heart development employing northern hybridization and RNase protection assay. The mRNAs for alpha1(II) and alpha1(IX) collagens were expressed transiently between embryonic days 10.5 and 14.5 in embryonic mouse heart. RNase protection assays revealed that for both transcripts the embryonic ("prechondrogenic") variants of the alternatively spliced mRNA isoforms dominated. Immunohistochemistry demonstrated that type IIA collagen and Sox9, its key transcriptional regulator, were expressed in the epithelial-mesenchymal areas of the developing heart, with partially overlapping patterns particularly in valvular and septal regions. In addition, Sox9 expression was detected widely in the developing heart. These observations support the hypothesis that cartilage collagens, especially the long isoform of type II collagen, participate in the morphogenesis of cardiac valves and septa.

Patterned collagen layers on polystyrene: direct probing using AFM in the adhesion mapping mode

Patterned collagen layers, showing a net-like structure with nanometer-scale dimensions, were obtained by collagen adsorption onto polystyrene (PS) followed by drying at low rate. These obtained surfaces were characterized and compared, on the one hand, to the PS substratum and, on the other hand, to uniform collagen layers obtained by adsorption onto PS and drying at high rate. In a first approach, atomic force microscope (AFM) images, X-ray photoelectron spectroscopy data and wetting measurements were compared. The results indicated that PS was present at the extreme surface in the holes of the collagen net. A second approach, allowing a direct and spatially-resolved probing of the surface chemistry, was based on the adhesion mapping mode of the AFM. The net-like structure observed on the AFM topographic images was also found in the adhesion map obtained in water: no or very low adhesion was found on collagen threads as observed on a uniform collagen layer; a higher adhesion was recorded in the holes of the net as observed on pure PS. This shows, in a direct manner, that the patterned surface is chemically heterogeneous, with PS domains present at the outermost surface in the holes of the collagen pattern, and that this is preserved under water.

Motile chondrocytes from newborn calf: migration properties and synthesis of collagen II

Objective: To determine whether differentiated chondrocytes are motile. Design: Calf articular chondrocytes isolated from six animals were cultured in spinner flasks and removed on days 3 and 7. Boyden chamber assays and time-lapse videomicroscopy were performed to monitor and quantify cell migration. A novel method for selectively harvesting and metabolically labeling the migrated cells was developed, based on cell movement to the underside of the Boyden chamber membranes. The 3H-collagen synthesized by these cells was purified and analyzed by SDS-PAGE and autoradiography either before or after cyanogen bromide cleavage. Results: In Boyden chambers, locomotion of day 3 chondrocytes on fibronectin-coated membranes was ∼3-fold higher than on bovine serum albumin-coated controls (39±15 vs 12±8 cells/mm 2, respectively ( P=0.005)). Insulin-like growth factor-I (IGF-I, 10 ng/ml) was chemotactic, increasing motility to 87±16 cells/mm − (difference from fibronectin alone: P=0.0003). A similar response was observed for day 7 cells, but IGF-I activation was not as pronounced ( P=0.055). The collagen patterns produced by the migrated cells closely resembled those of standard collagen type II, without any evidence of collagen I production. In videotracking experiments, motile cells attached on fibronectin exhibited typical lamellipodia and filopodia, and ∼30% of attached cells were motile (speed >1 μm/h and directional persistence >1 h). Typical cell path lengths were 30–50 μm, substantially greater than a full cell length displacement. Conclusion: A population of well-differentiated chondrocytes capable of matrix (COL II) synthesis are motile in vitro. This original finding opens new avenues to study the potential of motile cells for cartilage repair.

Differential expression of type XII collagen in developing chicken metatarsal tendons.

Type XII collagen is a fibril-associated collagen with multiple functional domains. The purpose of this work was to determine its role in regulating tendon matrix assembly. The temporal and spatial expression patterns of both collagen and mRNA were analysed in developing chicken metatarsal tendons using immunofluorescence microscopy, in situ hybridization and real-time quantitative PCR. Temporally, type XII collagen was present during all stages of development (day 14-hatch). However, spatially, type XII collagen expression shifted from the entire tendon at day 14, when the tendon is immature and fascicles are not well developed, to the interfacial matrix (endotendinium) associated with developing fascicles. This shift was obvious beginning at day 17, becoming prominent at day 19. Associated with this shift was a gradual decrease in type XII collagen reactivity in the tendon proper (non-sheath). By hatching, the reactivity was sequestered almost exclusively to the sheaths with some reactivity remaining at the fibroblast-matrix interface within the fascicle. In situ hybridization indicated that fibroblasts in the tendon expressed type XII collagen mRNA homogeneously at day 14. However, by hatching, when the tendon matures, type XII collagen is restricted primarily to the sheath cells. Quantitative PCR analyses, of NC3 splice variants, demonstrated highest expression levels for the short splice variant mRNA at days 14-17, followed by a significant decrease at day 19 with levels remaining constant to adult. Long variant mRNA expression was highest at day 14 then decreased and was constant from day 17 to adult. These changing patterns may be related to the spatial shift in type XII collagen expression to the sheaths. Differential temporal and spatial expression patterns indicate that type XII collagen functions to integrate the developing tendon matrices and fascicles into a functional unit.

Collagen type I expression in squamous cell carcinoma of the oral cavity

An analysis of the extracellular matrix at the invasive front of squamous cell carcinoma of the oral cavity may improve the understanding of tumour cell matrix interactions during malignancy growth. Alterations in collagen I expression may influence cellular invasion and metastasis. In this work, 23 cases of squamous cell carcinoma were submitted to the Anneroth's malignancy grading system. H. E. and sirius red staining were used. Immuno-histochemical expression of collagen type I protein was observed in different malignancy scores. As a result, it was observed that the extracellular matrix in squamous cell carcinoma of the oral cavity shows different patterns of collagen I expression in low and high scores of malignancy.

Modulatory effect of Coccinia indica on aortic collagen in streptozotocin-induced diabetic rats.

1. The effects of Coccinia indica, an indigenous plant used in Ayurvedic medicine in India, on aortic collagen content and its characteristics were assessed in streptozotocin (STZ) diabetic rats. 2. Rats were made diabetic with a single intraperitoneal injection of STZ (45 mg/kg). Blood glucose, hydroxyproline, collagen, extent of glycation, collagen-linked fluorescence, soluble pattern of pepsin-soluble collagen, shrinkage temperature, alpha/beta ratio of type I collagen and type I/type III collagen ratio were determined in rats treated with C. indica leaf extract (CLEt; 200 mg/kg for 45 days using an oral intragastric tube). 3. In diabetic rats, the collagen content, as well as the degree of cross-linking, was increased, as evidenced by increased shrinkage temperature and decreased pepsin solubility. The alpha/beta ratio of type I collagen and the type I/type III collagen ratio of pepsin-soluble collagen were significantly decreased in STZ diabetic rats. 4. In conclusion, administration of CLEt for 45 days to STZ diabetic rats significantly reduced the accumulation and cross-linking of collagen. The effects of C. indica (collagen content 23.87 +/- 1.52 mg/100 mg tissue (t value = 6.80), extent of cross-linking 0.893 +/- 0.072 mg hydroxyproline/100 mg tissue (t value = 9.0)) were comparable with those of glibenclamide (collagen content 26.18 +/- 1.65 mg/100 mg tissue (t value = 4.58), extent of cross-linking 0.787 +/- 0.057 mg hydroxyproline/100 mg tissue (t value = 7.1)), a reference drug.

Comments onSynchrotron fibre diffraction identifies and locates foetal collagenous breast tissue associated with breast carcinomaby V. J. James (2002).J. Synchrotron Rad.9, 71–76

The paper by James (2002) raises several questions. In our opinion, the small-angle scattering data shown in the article do not support all the conclusions of the author. We have analyzed the data and arrived at interpretations that are quite obvious, but not as exciting as those offered by the author. The scales in Figs. 2 and 3 are not given, but those can be established by indexing the collagen pattern in the meridional direction. In Fig. 3(c), inside the bright ring, re ections 00l, with l = 5, 6, 7, 8, 9, 10, 11, 12, are identi®ed. Using d = 65 nm for the axial period of collagen ®brils, we calculate the bright ring to correspond to a period of 4.24 nm. The bright ring arises from the presence of fat. The pattern is very typical for breast tissue samples, as seen in our Fig. 1 here, where azimuthal averages of SAXS patterns from fat and collagen are shown. The period giving rise to the fat peak is 4.26 nm, and in the two-dimensional pattern the intensity of the fat ring is uniform. The characteristic ring in the fat pattern is strong and narrow, like the ring in Fig. 3(c), and not diffuse and barely discernible as in Fig. 3(a). Triglyceride molecules in the `tuning fork' conformation are about 4 nm long, and they are arranged parallel to each other building layers, which are typically 4.25 nm thick. Layers form stacks, and this regular packing gives rise to the diffraction peak. It is obvious that the author has misinterpreted her data by assigning the 32.1 nm period to the fat ring. The right-hand side of Fig. 2 is actually taken from a previous publication (James et al., 1998). It shows several maxima, which correspond to the cylindrical ®brils and are described by the Bessel function J1 of the ®rst kind. There is also some ®ne structure, which the author claims to arise from the 353 nm periodicity of the `chicken wire' lattice associated with collagen IV. The intensity variations in Fig. 2(b) are very sharp, and, if they are due to diffraction from a lattice, the lattice must be extended and regular like in a para-crystal. We are not saying that such a lattice does not exist, but Figs. 2(b) and 2(c) cannot be used as evidence for the existence of the lattice. It is much more likely that the author has managed to index the noise of the SAXS pattern. In Fig. 1 here the collagen peaks are weak but clearly visible even though logarithmic scales are used. The strong peak in the fat pattern arises from layers of triglyceride molecules, and the same peak is visible in the scattering pattern of a collagen-containing sample. The intensities have been re-scaled for a better separation of the curves.

Utility of MMP-1, p53, E-cadherin, and collagen IV immunohistochemical stains in the differential diagnosis of adenomas with misplaced epithelium versus adenomas with invasive adenocarcinoma.

Adenomas with misplaced epithelium in the submucosa of the polyp stalk ("pseudoinvasion") may be difficult to distinguish from adenomas that harbor invasive adenocarcinoma by morphologic analysis. Recently, several epithelial and stromal proteins, such as matrix metalloproteinase-1 (MMP-1), p53, E-cadherin, and collagen IV, have been shown to be altered in colonic adenocarcinomas in comparison with adenomas and normal colonic mucosa. Therefore, the purpose of this study was to evaluate the diagnostic use of several epithelial (p53, E-cadherin) and stromal (MMP-1, collagen IV) markers in distinguishing adenomas with misplaced epithelium from those with invasive adenocarcinoma. Routinely processed polypectomy specimens from 23 patients with an adenoma with misplaced epithelium (male/female ratio 12/11; mean age 65 years) and 23 patients with an adenocarcinoma arising in an adenoma (male/female ratio 13/10; mean age 63 years) were immunohistochemically stained (avidin-biotin complex method) for monoclonal antibodies to MMP-1 (epithelial and stromal cell collagenase), p53 (tumor suppression gene), E-cadherin (intercellular adhesion protein), and collagen IV (basement membrane collagen component), and the results were compared between the two polyp groups. Where appropriate, immunopositivity was evaluated in the epithelium (MMP-1, p53, E-cadherin), stroma (MMP-1), and/or basement membrane (collagen IV). Cases were considered positive if an increase (MMP-1, p53) or decrease (E-cadherin, collagen IV) in either the intensity or proportion of cells staining was noted in the submucosal epithelial component compared with the intramucosal portion of the polyp head for each individual polyp. In adenomas with invasive adenocarcinoma, MMP-1 staining of the stroma surrounding submucosal epithelium and p53 nuclear staining within the epithelium were increased in 21 (91%) and 14 (61%) cases, respectively, whereas decreased or discontinuous E-cadherin and collagen IV staining was noted in 15 (65%) and 22 (96%) cases, respectively. All these values were significantly different (p < 0.005) from those observed in adenomas with misplaced epithelium [MMP-1, 11 of 23 (48%); p53, 1 of 23 (4%); E-cadherin, 0 of 23 (0%); collagen IV, 0/23 (0%)]. Furthermore, in three diagnostically difficult cases that contained foci of misplaced epithelium with high-grade dysplasia, the immunohistochemical results confirmed the impression that the lesions represented epithelial misplacement rather than invasive adenocarcinoma. In conclusion, the degree and/or pattern of MMP-1, p53, E-cadherin, and collagen IV staining in the submucosal epithelial elements in comparison with the intramucosal adenomatous tissue may help distinguish adenomas with misplaced epithelium from those with invasive adenocarcinoma.