Use of an anionic collagen matrix made from bovine intestinal serosa for in vivo repair of cranial defects.

Polymeric biomaterials composed of extracellular matrix components possess osteoconductive capacity that is essential for bone healing. The presence of collagen and the ability to undergo physicochemical modifications render these materials a suitable alternative in bone regenerative therapies. The objective of this study was to evaluate the osteogenic capacity of collagen-based matrices (native and anionic after alkaline hydrolysis) made from bovine intestinal serosa (MBIS). Twenty-five animals underwent surgery to create a cranial defect to be filled with native and anionic collagen matrixes, mmineralized and non mineralized. The animals were killed painlessly 6 weeks after surgery and samples of the wound area were submitted to routine histology and morphometric analysis. In the surgical area there was new bone formation projecting from the margins to the center of the defect. More marked bone neoformation occurred in the anionic matrices groups in such a way that permitted union of the opposite margins of the bone defect. The newly formed bone matrix exhibited good optical density of type I collagen fibers. Immunoexpression of osteocalcin by osteocytes was observed in the newly formed bone. Morphometric analysis showed a greater bone volume in the groups receiving the anionic matrices compared to the native membranes. Mineralization of the biomaterial did not increase its osteoregenerative capacity. In conclusion, the anionic matrix exhibits osteoregenerative capacity and is suitable for bone reconstruction therapies.

Cholera toxin B subunit-binding and ganglioside GM1 immuno-expression are not necessarily correlated in human salivary glands.

OBJECTIVE: To determine and compare the presence and in situ localization of the glycosphingolipid ganglioside GM1 in human salivary glands using the biomarkers for GM1: cholera toxin and antibodies against GM1. MATERIALS AND METHODS: Immunohistochemical analyses were performed on sections of adult human submandibular, parotid and palatinal glands using cholera toxin sub-unit B and two polyclonal antibodies against ganglioside GM1 as biomarkers. RESULTS: Immunofluorescence microscopy showed that the toxin and antibodies were co-localized in some acini but not in others. The cholera toxin mainly reacted with the cell membranes of the mucous acini in the submandibular gland, while incubation with the antibody against GM1 gave rise to a staining of the cytoplasm. The cytoplasm in some secretory acinar cells in the parotid gland was stained by the cholera toxin, whereas only small spots on the plasma membranes reacted with anti-GM1. The plasma membranes in the parotid excretory ducts appeared to react to anti-GM1, but not to cholera toxin. CONCLUSIONS: Cholera toxin induces the expression of ion channels and carriers in the small intestine and increases the production of secretory mucins. Although their mutual immunohistochemical localization may differ, both cholera toxin and ganglioside GM1 are present in the mucin-producing acini from salivary glands. This could point to a relationship between ganglioside expression and production of salivary mucins.

Epithelioid lesions of the serosa.

CONTEXT: Diagnosing epithelioid serosal lesions remains a challenge because numerous different processes-primary or secondary, benign or malignant-occur in body cavities, some of which are very rare. OBJECTIVES: To review the newest literature and to describe the morphologic criteria and immunohistochemical markers that are useful for distinguishing epithelioid serosal lesions. DATA SOURCES: Previously published literature concentrating on the newest research findings. Earlier reviews are principally referred to for established diagnostic criteria. CONCLUSIONS: Immunohistochemistry with a panel of antibodies has made the diagnosis of epithelioid serosal lesions very reliable. When deciding on antibodies used in differential diagnosis, it is important to consider tumor location, clinical and radiologic information, and morphologic features. Immunohistochemistry is less useful in the differential diagnosis of benign versus malignant mesothelial lesions. The diagnosis of benign versus malignant mesothelial proliferations still relies on the histologic criteria of invasion.

Determination of main low molecular weight antioxidants in urinary bladder wall using HPLC with electrochemical detector.

The aim of the present work was to develop validated HPLC method using electrochemical detector for simultaneous detection of low molecular weight antioxidants (LMWA) in urinary bladder. Furthermore, the method was applied to study the distribution of LMWA in urinary bladder wall. The ascorbic acid (AA), glutathione in reduced (GSH) and oxidized (GSSG) form and uric acid (UA) were resolved by isocratic elution from C18 reversed-phase column. The bladder tissue sample preparation involved extraction with meta-phosphoric acid solution for LMWA stabilization. The AA, GSH and UA tissue peak was identified by different approaches. The obtained method validation parameters were in acceptable range: intra-day precision (<4.4%), intra-day accuracy (<8.4%), inter-day precision (<9.4%) and inter-day accuracy (<15.6%). Additionally, the method provided good linearity (r2>0.99) and recoveries (98.9-112.6%). The distribution of LMWA in urinary bladder was determined by measuring their concentration in bladder wall layers: urothelium, lamina propria, muscularis and serosa. The validated method was able to quantify the reduced form of all three LMWA in all four bladder wall layers. The LMWA concentrations were decreasing from urothelium to serosa except of UA. The developed HPLC method with electrochemical detection of LMWA is simple, fast and can be used for simultaneous quantification of LMWA in tissues, which contain low concentrations of antioxidants.

Seromucous cells in human sublingual glands: examination by immunocytochemistry of lysozyme.

This study examined the occurrence and morphological features of serous-type cells in human sublingual gland, using immunocytochemistry for lysozyme. Lysozyme-positive cells usually formed demilunes and occasionally their own acini. They were also found among cells of an intercalated duct and in its immature acinus consisting of a small number of secretory cells. All these serous cells could be classified as seromucous cells because they simultaneously revealed reactivity for mucus, i.e., a periodic acid-Schiff (PAS) and a periodic acid-thiocarbohydrazide-silver proteinate (PA-TCHSP) reaction under the light- and electron-microscope, respectively. Immunogold labeling of lysozyme in the seromucous cells was distributed on variously sized secretory granules. These usually possessed a single electron-dense spherule in an electron-lucent matrix, while granules of a homogenous structure were also present. Lysozyme-positive cells filled with large, lucent secretory granules could hardly be morphologically distinguished from the lysozyme-negative mucous cells; they corresponded to "intermediate" cells designated under the light microscope. All "immature" secretory cells with only a few secretory granules were also lysozyme-positive seromucous cells. The present study demonstrated that the seromucous cells in the human sublingual glands conform closely with those in the human labial glands (MIYAZAKI et. al., 1998).

Functional and molecular characterization of an anion exchanger in airway serous epithelial cells.

Serous cells secrete Cl(-) and HCO(3)(-) and play an important role in airway function. Recent studies suggest that a Cl(-)/HCO(3)(-) anion exchanger (AE) may contribute to Cl(-) secretion by airway epithelial cells. However, the molecular identity, the cellular location, and the contribution of AEs to Cl(-) secretion in serous epithelial cells in tracheal submucosal glands are unknown. The goal of the present study was to determine the molecular identity, the cellular location, and the role of AEs in the function of serous epithelial cells. To this end, Calu-3 cells, a human airway cell line with a serous-cell phenotype, were studied by RT-PCR, immunoblot, and electrophysiological analysis to examine the role of AEs in Cl(-) secretion. In addition, the subcellular location of AE proteins was examined by immunofluorescence microscopy. Calu-3 cells expressed mRNA and protein for AE2 as determined by RT-PCR and Western blot analysis, respectively. Immunofluorescence microscopy identified AE2 in the basolateral membrane of Calu-3 cells in culture and rat tracheal serous cells in situ. In Cl(-)/HCO(3)(-)/Na(+)-containing media, the 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP)-stimulated short-circuit anion current (I(sc)) was reduced by basolateral but not by apical application of 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (50 microM) and 4, 4'-dinitrostilbene-2,2'-disulfonic acid [DNDS (500 microM)], inhibitors of AEs. In the absence of Na(+) in the bath solutions, to eliminate the contributions of the Na(+)/HCO(3)(-) and Na(+)/K(+)/2Cl(-) cotransporters to I(sc), CPT-cAMP stimulated a small DNDS-sensitive I(sc). Taken together with previous studies, these observations suggest that a small component of cAMP-stimulated I(sc) across serous cells may be referable to Cl(-) secretion and that uptake of Cl(-) across the basolateral membrane may be mediated by AE2.

Evaluation of histochemical methods for the detection of intracytoplasmic mucin in serous effusions.

We have studied 25 serous effusions containing definitive morphologic evidence of adenocarcinoma to evaluate the ability of two mucin stains (Mayer's mucicarmine, D-PAS) to detect intracytoplasmic mucin in both cytologic (cytospin) and corresponding histological (cell block) preparations. Mucicarmine stain was positive in six of 25 (24%) cytospins and 13 of 25 (52%) cell blocks. D-PAS was positive in 19 of 25 (76%) cytospins and 20 of 25 (80%) cell blocks. Eight cases were identified which showed mucicarmine positivity in the cell block but not the corresponding cytospin; prolonging incubation time resulted in a positive mucicarmine in cytospin preparations for seven of these cases. We conclude that: (i) D-PAS is a more sensitive stain for the detection of intracytoplasmic mucin in all preparations; (ii) mucicarmine shows preferential staining for cell blocks; (iii) alterations in the staining protocol may permit mucin detection by mucicarmine staining in cytologic preparations in a significant number of cases.

Isolation and molecular characterization of serous and mucous gland cells of the porcine airways.

Secretory cells of the glands of the airways play important roles in the pathogenesis of several diseases. Little, however, is known about the molecular biology of these cells. Here we describe a procedure for the separation of serous and mucous gland cells and the isolation of genes specifically expressed in these cells. Mucosal tissue was prepared from porcine large airways. Following enzymatic digestion, the cell types were separated by discontinuous Percoll density gradient centrifugation. Cell purity was analyzed by electron microscopy. The cell fractions contained between 75 and 85% mucous and serous cells, respectively. To isolate cell type-specific genes, poly(A)+ RNA was isolated from serous and mucous cell fractions, reverse transcribed and used for differential display polymerase chain reaction (PCR). Out of about a total of 1,700 PCR products identified in horizontal polyacrylamide gels, most bands were found to be common to both cell fractions, indicating that the transcript patterns in cells from both fractions are very similar. Eighteen PCR products, however, were consistently distinct in the two cell fractions, with eight products present only in RNA from the mucous cell fraction and 10 PCR products present only in RNA from the serous cell fraction. Dot-blot analysis of mRNA of serous and mucous cells proved the cell type-specific expression of nine PCR products. Northern blot analysis detected single transcripts for each PCR product. The development of a simple cell separation procedure for secretory cells of the airways, combined with the ability to isolate numerous cell type-specific marker genes, should facilitate the molecular understanding of secretory cells of the airways.

Control of germ-band retraction in Drosophila by the zinc-finger protein HINDSIGHT.

Drosophila embryos lacking hindsight gene function have a normal body plan and undergo normal germ-band extension. However, they fail to retract their germ bands. hindsight encodes a large nuclear protein of 1920 amino acids that contains fourteen C2H2-type zinc fingers, and glutamine-rich and proline-rich domains, suggesting that it functions as a transcription factor. Initial embryonic expression of hindsight RNA and protein occurs in the endoderm (midgut) and extraembryonic membrane (amnioserosa) prior to germ-band extension and continues in these tissues beyond the completion of germ-band retraction. Expression also occurs in the developing tracheal system, central and peripheral nervous systems, and the ureter of the Malpighian tubules. Strikingly, hindsight is not expressed in the epidermal ectoderm which is the tissue that undergoes the cell shape changes and movements during germ-band retraction. The embryonic midgut can be eliminated without affecting germ-band retraction. However, elimination of the amnioserosa results in the failure of germ-band retraction, implicating amnioserosal expression of hindsight as crucial for this process. Ubiquitous expression of hindsight in the early embryo rescues germ-band retraction without producing dominant gain-of-function defects, suggesting that hindsight's role in germ-band retraction is permissive rather than instructive. Previous analyses have shown that hindsight is required for maintenance of the differentiated amnioserosa (Frank, L. C. and Rushlow, C. (1996) Development 122, 1343-1352). Two classes of models are consistent with the present data. First, hindsight's function in germ-band retraction may be limited to maintenance of the amnioserosa which then plays a physical role in the retraction process through contact with cells of the epidermal ectoderm. Second, hindsight might function both to maintain the amnioserosa and to regulate chemical signaling from the amnioserosa to the epidermal ectoderm, thus coordinating the cell shape changes and movements that drive germ-band retraction.

Proliferation of submesothelial mesenchymal cells during early phase of serosal thickening in the rabbit bladder is accompanied by transient keratin 18 expression.

Partial outlet obstruction of the rabbit bladder induces serosal thickening and smooth muscle (SM) hypertrophy. Within thickened serosa, submesothelial (mesenchymal) cells differentiate into SM cells after 30 days of obstruction[S. Buoro et al. Lab. Invest. 69, 589-602, 1993]. Here, we show that submesothelial cells transiently express keratin (K) 18 but not K8 soon after obstruction. We investigated a possible relationship between keratin expression and cell proliferation/differentiation in vivo and in vitro. The results of this study indicate that expression of K18 is spatiotemporally related to the pattern of cell proliferation with respect to the localization of an elastic membrane which divides the thickened serosa into an "extrinsic" and an "intrinsic" region. Moreover, K18 is not present in bladder mesenchyma during early development, indicating that its expression in the adult is not attributable to a dedifferentiation process. However, simultaneous K18, K8, and desmoplakin (DP) expression can be induced in normal and thickened serosa upon treatment with bromo-deoxyuridine. Our results indicate that K18 is a marker of proliferating mesenchymal cells in rabbit serosa, whereas the combined expression of K18, K8, and DP might be related to the hypothesized alterations in the stability of gene expression. A model is proposed in which keratin-containing submesothelial cells can act as a "transit" cell phenotype involved in both regenerating mesothelial cells and formation of SM cells.

Serosal balls detected immunocytochemically in peritoneal lavage obtained during surgery.

Using the immunoperoxidase technique, we studied "serosal balls," which have features resembling those of cells from primary and metastatic tumors, and may thus complicate cytodiagnosis. Serosal balls were detected in 32 (18%) of 174 peritoneal washings. The balls consisted of oval clusters of cells in solid masses surrounded by flattened cells. The interior of the serosal balls was stained green with Papanicolaou method, showing the presence of homogeneous amorphous material, sometimes stained in a filamentous pattern. Almost all serosal balls were stained immunocytochemically for both keratin and vimentin. The interior was stained with antibodies against collagen types I and III. Therefore, these balls were fragments of serous membrane, and contained fibrous tissue and mesothelial cells.

Morphological and histochemical study of human submucosal laryngeal glands.

BACKGROUND: The respiratory submucosal glands are a major source of secretions in the airway. Human submucosal laryngeal glands have been scarcely studied, with no works existing about their ultrastructure and histochemistry. METHODS: Samples of epiglottis, ventricle, false vocal folds and true vocal folds were fixed in 10% buffered formalin for histochemical study with conventional and carbohydrate lectin histochemistry. Other samples were fixed in 2.5% glutaraldehyde and conventionally processed for transmission electron microscopy. RESULTS: The human submucosal laryngeal glands are composed of serious tubules; mucous tubules; collector duct; and final portion of this duct. The serous cells showed sialosulphomucins and affinity for WGA and Con-A lectins. With a previous treatment with neuraminidase, they also labelled with PNA. The mucous cells contained sialosulphomucins and showed affinity for WGA and DBA lectins in the samples proceeding from blood group A, and for WGA, UEA-I and LTA with those from blood group O. Ultrastructurally, the serous cells presented a wide variety of granules, cells in which seromucous granules predominated. The mucous cells presented larger-sized granules which were very electron-lucent. The collector duct was composed of mitochondria-rich cells and basal cells. A cell which we have termed "intermediate" was identified in the transition zone between the mucous tubules and the collector duct, and in the final portion of the collector duct. It had morphological characteristics as if it were a transition between a goblet cell and collector duct cell. Some nerve endings with cholinergic and peptidergic vesicles were found among the myoepithelial cells. CONCLUSIONS: These glands presented some histological differences from the bronchial glands, the mucous secretion was related to the blood group antigens, and the serous cells showed a wide variability in their secretory granules, many of them being of a seromucous type.

Identification of decorin proteoglycan in bovine tracheal serous cells in culture and localization of decorin mRNA in situ.

Bovine tracheal submucosal gland serous cells in culture synthesize and secrete proteoglycans and not mucin glycoconjugates. We are interested in the characterization and role of these proteoglycans in airway secretions. The major [35S]methionine-labeled proteoglycan present is identified as the small chondroitin/dermatan sulfate proteoglycan decorin (PG II. PG40). Consistent with its identity as decorin this proteoglycan showed average apparent molecular weights of 75,000 to 130,000 with a core protein of an average, M(r) of about 40,000 and with glycosaminoglycan chains sensitive to chondroitinase ABC lyase of an average M(r) of about 25,000. These data were obtained from gel chromatographic and SDS-PAGE analyses. Northern blot analysis and partial amino acid sequencing of the purified protein further confirmed its identity as decorin. In situ hybridization studies using a decorin riboprobe revealed no expression of decorin in the surface epithelium and only low levels of expression in submucosal gland epithelial cells of bovine tracheal tissue. However, high levels of expression were localized to cells which are peripheral to tracheal submucosal gland epithelial cells and which contact with the extracellular matrix.

Lamellar bodies in synoviocytes, mesothelium and specific epithelia as possible site of auto-antigen in rheumatoid disease.

Intracytoplasmic lamellar organelles identical in ultrastructure to surfactant-containing lamellar bodies found in type II pneumocytes, have been demonstrated in other tissues, in synoviocytes and mesothelial cells, in a distribution pattern which reflects the systemic expression of rheumatoid disease. Antibodies raised against surfactant protein A (SP-A), exhibit a ranking of tissue reactivity in area, intensity and density of cells which also parallels the frequency and degree of pathological involvement characteristic of rheumatoid disease, showing in ascending order of immunopositivity, lacrymal and salivary epithelia, pulmonary parenchyma, mesothelium and synoviocytes. Maximal tissue reactivity to anti-SP-A antibodies was found in the synovium of 55 rheumatoid patients exhibiting classical histopathological appearances of RA, in a pattern of immunostaining identical to that obtained with ML30, an antibody to mycobacterial heat shock protein 65kDa which, in turn, cross-reacted with SP-A in dot blot testing.

Stromal distribution of CA 19-9 as a predictor of lymph node metastases in gallbladder cancer without serosal invasion.

Gallbladder cancer without serosal invasion is often diagnosed during the pathologic examination of a gallbladder removed for presumed benign disease. The role of reoperative procedures in these patients is not well established, making it important to define prognostic factors that predict cancer spread. To determine if the pattern of immunohistochemical staining with a monoclonal antibody against CA 19-9 could predict lymph node spread of cancer, 23 patients with gallbladder cancer without serosal invasion were reviewed. CA 19-9 reaction was present in pathology specimens from all 23 patients. Twelve samples (52.2%) showed CA 19-9 in the stroma adjacent to cancer cells, while the remaining 11 did not show any stromal staining against CA 19-9. Lymph node development was found in only 2 (18.2%) of these 11 patients in the nonstromal staining group and 9 (75%) of the 12 patients in the stromal staining group. As these differences were statistically significant, we conclude that immunohistochemical localization of CA 19-9 in gallbladder cancer may be useful in predicting the presence or absence of lymph node involvement, and in developing a rational approach for a reoperative procedure.

The effect of vesical outlet obstruction on the protein secondary structure of the mucosa and serosa in rabbit bladder wall.

The biochemical composition of physiologically moist mucosa and serosa of rabbit bladder before and after bladder outlet obstruction was determined by means of FT-IR spectroscopy with the ATR method and second-derivative analysis. A predominantly beta-sheet structure was found in the amide I band for mucosa and serosa before and after obstruction, but the random coil structure increased in both obstructed bladder samples. However, the major beta-sheet structure associated with some alpha-helical structure in the amide II band of mucosa and serosa for non-obstructed bladder changed into a predominantly alpha-helical structure after bladder obstruction. The obstructed bladder serosa was more pronounced. The amount of glycoproteins doubled in the obstructed bladder serosa, but did not change in the bladder mucosa.

Epithelial markers to detect occult microinvasion in serous ovarian tumors.

Ovarian serous lesions with severe cytological and architectural atypia without obvious destructive stromal invasion may be diagnosed as serous borderline malignant tumor or as serous cystadenocarcinoma, depending on the criteria that individual pathologists use. The recent introduction of the diagnosis "serous borderline tumors with stromal microinvasion" seems an important improvement for the accuracy of the diagnosis of serous ovarian tumors. The aim of this study was to determine if immunohistochemical epithelial markers could help to detect stromal microinvasion in serous ovarian tumors and to compare these findings with the occurrence of "eosinophilic metaplastic" cells. Therefore, we studied the presence of eosinophilic metaplastic cells. Three immunohistochemical epithelial markers were applied in a group of 42 borderline and invasive serous tumors. The histopathologic diagnosis of the tumors was established by a reference center for gynecologic pathology in the Netherlands. We found that "eosinophilic metaplastic" cells were a constant feature in the serous borderline tumor lesions both with and without microinvasion. The presence of these cells should therefore not be considered as pathognomonic for microinvasion. The three investigated antibodies against epithelial epitopes helped to detect microinvasion, with the monoclonal antikeratin (CAM5.2) the best of these antibodies. Serous tumors diagnosed as carcinoma with dubious invasion showed no evidence of microinvasion in 83% of cases. In 13% of serous borderline malignant tumors, microinvasion was detected by the antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)