Histological, Histomorphometrical, and Biomechanical Studies of Bone-Implanted Medical Devices: Hard Resin Embedding.

The growing incidence of degenerative musculoskeletal disorders as well as lifestyle changes has led to an increase in the surgical procedures involving implanted medical devices in orthopedics. When studying implant/tissue interface in hard materials (i.e., metals or dense plastics) and/or in large bone segments, the hard plastic embedding of the intact undecalcified tissue envelope with the implant in situ is needed. The aim of this work is to describe the advances and the possibilities of high-temperature methyl methacrylate (MMA) embedding for the histological, histomorphometrical, and biomechanical assessment of bone-implanted medical devices. Unlike routine techniques, undecalcified bone processing histology, using high-temperature MMA, requires a complex and precise sample processing methodology and the availability of sophisticated equipment and software for both sample preparation and analyses. MMA embedding permits the evaluation of biological responses to the presence of implanted medical devices without implant removal, allowing simultaneous qualitative and quantitative histological evaluation, both static and dynamic histomorphometry, and biomechanical analyses not possible with tissue decalcification. MMA embedding, despite being a demanding procedure, is still preferred to other kinds of resin-based embedding because of its peculiar characteristics, which allow the study of samples of big dimensions also implanted with hard materials without reducing the sample or removing the material. Dynamic measurements are allowed together with biomechanical investigations at the bone-biomaterial interface, obtaining a comprehensive and precise evaluation of the safety and effectiveness of medical devices for orthopedic regenerative, reconstructive, and reparative surgery.

Current Trends in the Evaluation of Osteochondral Lesion Treatments: Histology, Histomorphometry, and Biomechanics in Preclinical Models.

Osteochondral lesions (OCs) are typically of traumatic origins but are also caused by degenerative conditions, in primis osteoarthritis (OA). On the other side, OC lesions themselves, getting worse over time, can lead to OA, indicating that chondral and OC defects represent a risk factor for the onset of the pathology. Many animal models have been set up for years for the study of OC regeneration, being successfully employed to test different treatment strategies, from biomaterials and cells to physical and biological adjuvant therapies. These studies rely on a plethora of post-explant investigations ranging from histological and histomorphometric analyses to biomechanical ones. The present review aims to analyze the methods employed for the evaluation of OC treatments in each animal model by screening literature data within the last 10 years. According to the selected research criteria performed in two databases, 60 works were included. Data revealed that lapine (50% of studies) and ovine (23% of studies) models are predominant, and knee joints are the most used anatomical locations for creating OC defects. Analyses are mostly conducted on paraffin-embedded samples in order to perform histological/histomorphometric analyses by applying semiquantitative scoring systems and on fresh samples in order to perform biomechanical investigations by indentation tests on articular cartilage. Instead, a great heterogeneity is pointed out in terms of OC defect dimensions and animal's age. The choice of experimental times is generally adequate for the animal models adopted, although few studies adopt very long experimental times. Improvements in data reporting and in standardization of protocols would be desirable for a better comparison of results and for ethical reasons related to appropriate and successful animal experimentation.

Engineered exosomes: A new promise for the management of musculoskeletal diseases.

BACKGROUND: Exosomes are nanovesicles actively secreted by potentially all cell types, including tumour cells, with the primary role of extracellular systemic communication mediators, both at autocrine and paracrine levels, at short and long distances. Recently, different studies have used exosomes as a delivery system for a plethora of different molecules, such as drugs, microRNAs and proteins. This has been made possible thanks to the simplicity in exosomes engineering, their great stability and versatility for applications in oncology as well as in regenerative medicine. SCOPE OF REVIEW: The aim of this review is to provide information on the state-of-the-art and possible applications of engineered exosomes, both for cargo and specific cell-targeting, in different pathologies related to the musculoskeletal system. MAJOR CONCLUSIONS: The use of exosomes as therapeutic agents is rapidly evolving, different studies explore drug delivery with exosomes using different molecules, showing an enormous potential in various research fields such as oncology and regenerative medicine. GENERAL SIGNIFICANCE: However, despite the significant progress made by the different studies carried out, currently, the use of exosomes is not a therapeutic reality for the considerable difficulties to overcome.

Intestinal anti-transglutaminase 2 immunoglobulin A deposits in children at risk for coeliac disease (CD): data from the PreventCD study.

In coeliac disease (CD), anti-tissue transglutaminase 2 immunoglobulin (Ig)A antibodies (anti-TG2) are produced and deposited in the intestine. PreventCD (www.preventcd.com) is a European multi-centre study, which investigates the influence of infant nutrition and that of genetic, immunological and other environmental factors on the risk of developing CD. The aim of the current study was to evaluate the appearance of intestinal anti-TG2 deposits in very early intestinal biopsies from at-risk infants and their predictive value for villous atrophy. Sixty-five small bowel biopsies, performed in 62 children, were investigated for the presence of intestinal anti-TG2 extracellular IgA deposits by using double immunofluorescence. The biopsies were performed in the presence of elevated serum levels of CD-associated antibodies and/or symptoms suggesting disease. Deposits of anti-TG2 IgA were present in 53 of 53 CD patients and three of three potential CD patients. In potential CD patients, mucosal deposits showed a patchy distribution characterized by some areas completely negative, whereas active CD patients had uniformly present and evident mucosal deposits. Only one of six patients without CD (negative for serum anti-TG2 and with normal mucosa) had intestinal deposits with a patchy distribution and a weak staining. Two of the 53 CD patients received a definitive diagnosis of CD after a second or third biopsy; mucosal deposits of anti-TG2 IgA were evaluated in all samples. Before developing villous atrophy, both patients had anti-TG2 deposits in normal mucosal architecture, antibodies in one patient being absent in serum. We demonstrated that in CD the intestinal deposits of anti-TG2 are a constant presence and appear very early in the natural history of disease.

Chondroprotective activity of N-acetyl phenylalanine glucosamine derivative on knee joint structure and inflammation in a murine model of osteoarthritis.

OBJECTIVE: Osteoarthritis (OA), the most common chronic degenerative joint disease, is characterized by joint structure changes and inflammation, both mediated by the IκB kinase (IKK) signalosome complex. The ability of N-acetyl phenylalanine derivative (NAPA) to increase cartilage matrix components and to reduce inflammatory cytokines, inhibiting IKKα kinase activity, has been observed in vitro. The present study aims to further clarify the effect of NAPA in counteracting OA progression, in an in vivo mouse model after destabilization of the medial meniscus (DMM). DESIGN: 26 mice were divided into three groups: (1) DMM surgery without treatment; (2) DMM surgery treated after 2 weeks with one intra-articular injection of NAPA (2.5 mM) and (3) no DMM surgery. At the end of experimental times, both knee joints of the animals were analyzed through histology, histomorphometry, immunohistochemistry and microhardness of subchondral bone (SB) tests. RESULTS: The injection of NAPA significantly improved cartilage thickness (CT) and reduced Chambers and Mankin modified scores and fibrillation index (FI), with weaker MMP13, ADAMTS5, MMP10 and IKKα staining. The microhardness measurements did not shown statistically significant differences between the different groups. CONCLUSIONS: NAPA markedly improved the physical structure of articular cartilage while reducing catabolic enzymes, extracellular matrix (ECM) remodeling and IKKα expression, showing to be able to exert a chondroprotective activity in vivo.

Intestinal titres of anti-tissue transglutaminase 2 antibodies correlate positively with mucosal damage degree and inversely with gluten-free diet duration in coeliac disease.

It has always been known that anti-tissue transglutaminase 2 (anti-TG2) antibodies are produced in the small intestine. Their serum titres correlate with mucosal damage degree and decrease on a gluten-free diet (GFD). We aimed to correlate intestinal anti-TG2 antibodies levels with degree of mucosal damage and GFD duration. Thirty-four active, 71 potential and 24 CD patients on GFD for at least 2 years were enrolled. Anti-TG2 deposits were detected in intestinal biopsies by double immunofluorescence. Biopsies were cultured for 24 h with medium, and with gliadin peptic tryptic digest (PTG) or A-gliadin peptide 31-43 (P31-43). Anti-TG2 antibodies secreted into supernatants were measured by enzyme-linked immunosorbent assay (ELISA). All active CD patients secreted high titres of anti-TG2 antibodies into culture medium that increased with the worsening of mucosal injury (Spearman's r = 0·71; P < 0·0001). Seventy of 71 potential CD patients and 15 of 24 treated CD patients secreted low titres of anti-TG2 antibodies into supernatants, eight of nine negative treated patients being on GFD for more than 10 years. An inverse correlation between antibody titres and duration of GFD was found, (Spearman's r = -0·52; P < 0·01). All active, 53 of 71 potential and six of 24 treated, CD patients showed anti-TG2 mucosal deposits. Five of six positive treated CD patients had been on GFD for fewer than 6 years and were also positive for secreted anti-TG2. In treated patients, PTG/P31-43 was not able to induce secretion of anti-TG2 antibodies into culture medium. Measurement of anti-TG2 antibodies in biopsy supernatants proved to be more sensitive than detection by immunofluorescence to reveal their intestinal production. Intestinal antiTG2 antibodies titres correlated positively with the degree of mucosal damage and inversely with the duration of GFD.

Intestinal anti-tissue transglutaminase antibodies in potential coeliac disease.

Anti-tissue transglutaminase 2 (anti-TG2) antibodies are present in the serum of the great majority of untreated coeliac disease (CD) patients. They are produced and deposited in the small intestinal mucosa. Potential CD patients present serum anti-TG2 antibodies higher than cut-off, but a normal duodenal mucosa where mucosal deposits of anti-TG2 are not always detectable. The aim of our work was to investigate the presence of anti-TG2 intestinal antibodies in patients with potential CD, and identify the most sensitive test to detect them. Twelve active CD patients, 28 potential CD patients and 39 non-CD controls were enrolled. Biopsy fragments from all patients were analysed by double immunofluorescence to detect mucosal deposits of anti-TG2 antibodies. Fragments from the same subjects were also cultured for 24 h with medium in the presence or absence of gliadin peptides. Anti-TG2 autoantibodies secreted into supernatants were measured by enzyme-linked immunosorbent assay. All active CD, 68% of potential CD patients and 20% of non-CD controls showed mucosal deposits of immunoglobulin (Ig)A anti-TG2; at the same time 100, 96 and 8% of active CD, potential CD and non-CD control patients secreted these antibodies in culture supernatants, respectively. Our data showed that, to detect intestinal anti-TG2 antibodies, the measurement of antibodies secreted into culture supernatants has higher sensitivity and specificity (97·5 and 92·3%, respectively) than the detection of mucosal deposits (77·5 and 80·0%, respectively). The measurement of intestinal anti-TG2 antibodies may prove useful in clinical practice to predict evolution towards mucosal atrophy in potential coeliac patients and identify patients with gluten sensitivity.

High density of intraepithelial gammadelta lymphocytes and deposits of immunoglobulin (Ig)M anti-tissue transglutaminase antibodies in the jejunum of coeliac patients with IgA deficiency.

The diagnosis of coeliac disease (CD) represents a special challenge in selective immunoglobulin (Ig)A deficiency (IgAD). A high density of T cell receptor (TCR)gammadelta(+) intraepithelial lymphocytes (IELs) and intestinal IgA anti-tissue transglutaminase 2 (anti-TG2) antibody deposits are suggestive of CD. We analysed the density of TCRgammadelta(+) IELs and the deposition of IgM anti-TG2 antibodies in the jejunal mucosa of IgAD patients with and without CD. Immunohistochemical analyses for the number of CD3+ and TCRgammadelta(+) IELs and double immunofluorescence assay for IgM anti-TG2 antibody deposits were performed in biopsies from 25 children with IgAD (nine untreated CD, seven potential CD and nine without CD). Sixteen immunologically intact children without CD represented the controls. IgAD without CD had a higher number of CD3+ and TCRgammadelta(+) IELs than controls (P < 0.05), but lower than IgAD with CD (P < 0.01). No significant differences were noted between IgAD subjects without CD and those with potential CD. Furthermore, IgAD patients without CD showed a higher TCRgammadelta(+)/CD3+ ratio than the control group (P < 0.05), while the ratio was similar to subjects with CD and potential CD. Intestinal IgM anti-TG2 antibody deposits were present in six of seven of the IgAD patients with untreated CD, one of seven with potential CD and none of those without CD. Most of the patients with IgAD show immune activation in the jejunal mucosa. IgM anti-TG2 antibody deposits are present only in CD. Intestinal IgM anti-TG2 and immunohistochemical markers do not discriminate between IgAD and potential CD with IgAD. Therefore, the serum IgG CD-associated autoantibodies remains very important for the diagnosis of CD in IgAD.

An immunodominant DQ8 restricted gliadin peptide activates small intestinal immune response in in vitro cultured mucosa from HLA-DQ8 positive but not HLA-DQ8 negative coeliac patients.

BACKGROUND: Studies on intestinal T cell clones from the mucosa of patients with coeliac disease have led to the identification of immunogenic gliadin epitopes. One is HLA-DQ8 restricted, its recognition by T cells being increased by introduction of negatively charged residues operated by tissue transglutaminase. AIM: To test HLA-DQ8 restricted epitope in both native (QYPSGQGSFQPSQQNPQA) and deamidated (QYPSGEGSFQPSQENPQA) forms in an organ culture system of treated coeliac mucosa from HLA-DQ8 positive and HLA-DQ8 negative patients. PATIENTS AND METHODS: Jejunal biopsies obtained from 10 patients with coeliac disease (six HLA-DQ8 positive and four HLA-DQ8 negative) were cultured in vitro with a peptic-tryptic digest (PT) of gliadin, or with the native (peptide A) or deamidated (peptide B) peptide. Intraepithelial CD3(+) and lamina propria total CD25(+) and CD3(+)CD25(+) cells were counted, lamina propria intercellular adhesion molecule 1 (ICAM-1) expression was evaluated, as well as that of Fas molecules on epithelial cells. RESULTS: In HLA-DQ8 positive, but not in HLA-DQ8 negative, coeliacs the density of intraepithelial CD3(+) cells, lamina propria total CD25(+), and CD3(+)CD25(+) cells, as well as expression of ICAM-1 and Fas molecules were significantly increased in biopsies cultured with PT, peptide A, or peptide B compared with biopsies cultured in medium alone. CONCLUSION: These data show that the DQ8 restricted gliadin peptide is immunogenic only in the intestinal mucosa of HLA-DQ8 positive coeliac patients in both native and deamidated forms.

Anti-tissue transglutaminase antibodies from coeliac patients inhibit transglutaminase activity both in vitro and in situ.

BACKGROUND AND AIMS: Coeliac disease (CD) is a multifactorial disorder which has an autoimmune component characterised by the occurrence of disease specific autoreactive antibodies against the enzyme tissue transglutaminase (tTG). The aim of this study was to investigate whether binding of antibodies to the enzyme influences tTG activity. METHODS: tTG activity was assayed in the presence of immunoglobulin A (IgA) and immunoglobulin G (IgG) purified from the serum of coeliac patients, CUB 7402 (an anti-tTG mouse monoclonal antibody), and human anti-tTG monoclonal antibodies derived from both intestinal lymphocytes from three patients with CD and from peripheral blood lymphocytes from healthy subjects. For our studies we used calcium treated and untreated recombinant human tTG. Furthermore, the effects of antibodies were determined by immunohistochemical detection of tTG activity in sections of human umbilical cord. RESULTS: IgG and IgA from CD patients inhibited tTG activity in vitro in a dose dependent manner, with a different rate of inhibition among patients. The monoclonal antibody CUB 7402 and human monoclonal antibodies displayed a dose dependent inhibitory effect towards the catalytic activity of the enzyme, both in vitro and in situ. Preincubation of tTG with CaCl(2) caused loss of the inhibitory effect due to CUB 7402 but not that caused by human monoclonal antibodies. CONCLUSIONS: Purified CD IgA, IgG, as well as human anti-tTG monoclonal antibodies inhibited the enzymatic activity of human tTG both in vitro and in situ.

Immune response of the coeliac nasal mucosa to locally-instilled gliadin.

We previously demonstrated a specific gluten-induced response in the rectal mucosa of coeliac patients. In the present study, we have evaluated the immune response to local gliadin challenge in the nasal mucosa of coeliac patients preliminary to exploring the feasibility of immune modulation by the nasal route. The local response to gliadin was evaluated on non-invasive scrapings of nasal mucosa. Cells harvested from the nasal scrapings of 21 coeliac patients and 12 healthy controls were counted after immunohistochemical staining. Six hours after gliadin challenge, the total number of cells was increased in coeliacs but not in controls. The increase was due principally to lymphoid cells and granulocytes. CD3+ cells doubled after gliadin challenge, but not after albumin control challenge. There was a similar rise in CD25+ cells, whereas the number of ICAM-expressing cells did not increase significantly. In control subjects, both gliadin and albumin induced a moderate but not significant increase in total cell number. In conclusion, the gliadin antigen provokes a mild inflammatory response in coeliac nasal mucosa.

The first large population based twin study of coeliac disease.

BACKGROUND AND AIMS: The genetic load in coeliac disease has hitherto been inferred from case series or anecdotally referred twin pairs. We have evaluated the genetic component in coeliac disease by estimating the concordance rate for the disease among twin pairs in a large population based study. METHODS: The Italian Twin Registry was matched with the membership lists of a patient support group. Forty seven twin pairs were recruited and screened for antiendomysial (EMA) and antihuman-tissue transglutaminase (anti-tTG) antibodies; zygosity was verified by DNA fingerprinting and twins were typed for HLA class II DRB1 and DQB1 molecules. RESULTS: Concordance rates for coeliac disease differ significantly between monozygotic (MZ) (0.86 probandwise and 0.75 pairwise) and dizygotic (DZ) (0.20 probandwise and 0.11 pairwise) twins. This is the highest concordance so far reported for a multifactorial disease. A logistic regression model, adjusted for age, sex, number of shared HLA haplotypes, and zygosity, showed that genotypes DQA1*0501/DQB1*0201 and DQA1*0301/DQB1*0302 (encoding for heterodimers DQ2 and DQ8, respectively) conferred to the non-index twin a risk of contracting the disease of 3.3 and 1.4, respectively. The risk of being concordant for coeliac disease estimated for the non-index twin of MZ pairs was 17 (95% confidence interval 2.1-134), independent of the DQ at risk genotype. CONCLUSION: This study provides substantial evidence for a very strong genetic component in coeliac disease, which is only partially due to the HLA region.

Characterization of the inflammatory infiltrate in peptic oesophagitis.

BACKGROUND: The diagnosis of oesophagitis is mainly based on histology, but interpretation of endoscopic biopsies is often difficult. We performed immunohistochemical studies on oesophageal biopsies to see if better characterization of the inflammatory cell infiltrate would improve the accuracy of the histologic diagnosis of gastro-oesophageal reflux disease. METHODS: The study groups consisted of 40 consecutive children (mean age +/- SD: 79.6 +/- 5l.9 months; 20 boys) with gastro-oesophageal reflux disease and 7 symptomatic children (mean age +/- SD: 52.6 +/- 37.0 months; 3 boys) without gastro-oesophageal reflux disease. All patients underwent upper gastrointestinal endoscopy with oesophageal biopsies. The diagnosis of gastro-oesophageal reflux disease was established by conventional endoscopic and histologic criteria. In each mucosal biopsy specimen, the number of intraepithelial CD3+, CD25+ (IL2 receptor+), ICAM+, HLA-DR+ and mucosal mast cells were determined. RESULTS: Conventional histology was in close agreement with endoscopic findings (p<0.001) and reflected the clinical score even more than endoscopic findings. Conventional histology significantly correlated with each inflammatory immunohistochemical marker (<0.05 for each), but the markers were not predictive of symptom severity. Immunohistochemical markers were always abnormal in the gastro-oesophageal reflux disease patients, even in the mildest cases of oesophagitis. CONCLUSIONS: Although there is a good correlation between symptoms and histology, in a subset of patients, immunohistochemical studies appear useful in supporting the histological diagnosis of gastro-oesophageal reflux disease.

An immunocytochemical study of intrapancreatic ganglia, nerve fibres and neuroglandular junctions in Brockmann bodies of the tompot blenny (Blennius gattoruggine), a marine teleost.

The innervation of the Brockmann bodies in the teleost fish, Blennius gattoruggine, was studied using immunocytochemical techniques at both the light and electron microscopy levels. Islet innervation consisted of intrapancreatic ganglia, generally localized inside the rim of the exocrine tissue of the Brockmann bodies, in proximity to the islet, nerve fibres and nerve terminals with synaptic complexes. The intrapancreatic ganglia were of variable size, with different numbers of ganglionic cells, that appeared unipolar in section. The cell bodies showed immunoreactivity to galanin, oxytocin, peptide tyrosine tyrosine and glucagon. The extrinsic and intrinsic nerve fibres passed through the exocrine parenchyma and crossed the connectival septa and islet connectival sheath, penetrating into the islets, where they became increasingly thinner. They terminated on the endocrine cells with dilated nerve terminals. At least three types of terminals were detected, depending on the different vesicle content: peptidergic, cholinergic or adrenergic. They presented specialized synaptic structures, the neuroglandular junctions, some of which contained neurosecretory granules immunogold labelled by galanin antiserum. This new finding confirms the role of galanin as a neurotransmitter. This rich supply of innervation may be important in the regulation and integration of islet secretion.

Organ culture of rectal mucosa : in vitro challenge with gluten in celiac disease.

Celiac disease is sustained by an immunological process that mainly affects the jejunal mucosa (1). Nonetheless, jejunum is not the only site of the gastrointestinal tract that is involved in celiac disease. In recent years, Ensari and colleagues (2,3), by using immunohistochemical analysis and computerized image analysis for numerical quantitation, have significantly contributed to a definitive and clear demonstration of a celiac disease-associated "proctitis," and its gluten dependence. Morphometry has shown increased populations of plasma cells, lymphocytes, and mast cells in the rectal mucosa of untreated patients, with these changes being reverted, with the sole exception of mast cells, by dietary treatment (2). The immunohistochemical approach has demonstrated highly significant increases in CD3(+) and γδ(+) lymphocytes within both the lamina propria and the epithelium. Mononuclear cells, both lymphocytes (CD3(+)) and macrophages (CD68(+)) expressing interleukin-2 (IL-2) receptors (CD25(+)), have been found to be increased in the lamina propria, usually immediately below the basal lamina. Enterocytes have been noted to be positive for major histocompatibility complex class II display, a pattern usually absent in normal colon. Furthermore, increased expression of vascular cell adhesion molecule-1 (VCAM-1) molecules in the rectal mucosa of untreated, compared to either treated celiac rectum or control mucosae, has been reported (3). As a whole, these data suggest, analogously to jeunum, an ongoing T-cell-dependent, cell-mediated immune response in the rectal mucosa.

Morphological basis of the interactions between endocrine cell types in the pancreatic islets of the teleost, Blennius gattoruggine.

The endocrine pancreas of the teleost fish Blennius gattoruggine was studied by immunochemistry using both light and electron microscopy. Generally, one large Brockmann body, along with intermediate and small islets, was found. Cells immunoreactive (IR) to anti-insulin (B), anti-glucagon (A) anti-somatostatin (D) anti-pancreatic polypeptide and anti-PYY sera were detected with B cells located at the center of the islet and the other cell types forming a peripheral mantle. The B-cell cytoplasm showed rows of microtubules close to the secretory granules and perpendicular to the plasmalemma. The ultrathin section images revealed exocytotic and endocytotic features, and the presence of intercellular gap junctions between the plasmalemma of contiguous cells, suggesting intercellular routes of communication, e.g. via autocrine and/or paracrine mechanism. These features were observed in all of the cell types, and were abundant in D cells. D cells were particularly numerous in the islets and were disposed close to A and B cells, as observed in other teleost species. The most peripheral B cells, in closer contact with D cells than the central ones, appeared strongly immunolabeled, perhaps owing to the inhibitory action of somatostatin. Some D cells exhibited a long protrusion directed towards the center of the islet. In view of their cytological characteristics and their secretion, D cells might have an important role in the modulation of A and B-cell secretion in an endocrine and/or paracrine fashion.

Maintenance of the differentiated type II cell phenotype by culture with an apical air surface.

The study of differentiated functions of alveolar type II cells has been hampered because of the lack of good in vitro systems. We report that culture of type II cells on collagen gels with an apical surface exposed to air promotes expression of differentiated type II cell characteristics. Cells cultured in this manner are cuboidal, contain lamellar bodies, and produce tubular myelin; in addition, they secrete phosphatidylcholine in response to exogenous ATP. Cultures contain mRNA for surfactant proteins A, B, and C and surfactant proteins A, B, and D. In contrast, when type II cells are cultured with an apical surface exposed to liquid rather than to air, the cells are squamous, do not express surfactant proteins or their respective mRNA, and do not contain lamellar bodies or produce tubular myelin. Type II cells cultured on plastic for 7 days, which no longer express mRNA for surfactant proteins, can be induced to express these mRNA by changing culture conditions to that of an air surface. The culture system described in this paper should be useful for studies of surfactant metabolism, regulation of alveolar epithelial phenotypic expression, and the processing of transiently expressed transgenes.

Islets and diffuse endocrine component in the pancreas of three red frogs species: relationships between endocrine and exocrine tissue.

The endocrine pancreas of three red frogs was studied immunohistochemically. It consisted of islets and diffuse endocrine cells. The islets showed a mammalian-like arrangement with a central core of B cells and a peripheral mantle of A/PP cells. A few D and VIP cells were also present. Several regulatory peptides were co-localized in the same endocrine cells by consecutive sections and double-labeling studies. The A/PP cells were formed by subpopulations of cells showing various types of immunoreactivity and varying degrees of immunolabeling. Generally, glucagon/pancreatic polypeptide, glucagon/pancreatic polypeptide/peptide tyrosine tyrosine and glucagon/pancreatic polypeptide/neuropeptide tyrosine immunoreactivities were present in the islets and in the endocrine cells scattered throughout the exocrine parenchyma (the diffuse component). Some specimens, mainly belonging to Rana dalmatina, showed evident periinsular halos around the islets. The diffuse component was abundant, and mainly contained A/PP cells. It formed a net across the exocrine parenchyma; its interrelationship with the latter might occur by a paracrine mechanism.

[Acute gangrenous appendicitis in incarcerated inguinal hernia. A case report]. / Appendicite acuta gangrenosa in ernia inguinale strozzata. Descrizione di un caso.

A case of acute gangrenous appendicitis in the sac of right inguinal hernia in a 77 year-old man is described. This condition is extremely uncommon; it is very difficult to establish a correct diagnosis preoperatively; a high mortality rate is due to extensive peritoneal contamination; primary hernia repair is indicated.

Angioma serpiginoso en dos niños / Angioma serpiginosum in two children

El angioma serpiginoso es una lesión de tipo nevoide, poco frecuente, benigna, progresiva y asintomática, de aparición en edad temprana, que se caracteriza por máculas rojizas a violáceas que siguen un trayecto serpiginoso correspondiente a dilataciones capilares en la dermis papilar y subpapilar. Presentamos dos pacientes, una niña de 6 años y un varón de 5 años, cuyas características clínicas e histopatológicas corresponden a esta entidad