Gene electrotransfer of IL-2 and IL-12 plasmids effectively eradicated murine B16.F10 melanoma.

Gene therapy has become an important approach for treating cancer, and electroporation represents a technology for introducing therapeutic genes into a cell. An example of cancer gene therapy relying on gene electrotransfer is the use of immunomodulatory cytokines, such as interleukin 2 (IL-2) and 12 (IL-12), which directly stimulate immune cells at the tumour site. The aim of our study was to determine the effects of gene electrotransfer with two plasmids encoding IL-2 and IL-12 in vitro and in vivo. Two different pulse protocols, known as EP1 (600 V/cm, 5 ms, 1 Hz, 8 pulses) and EP2 (1300 V/cm, 100 µs, 1 Hz, 8 pulses), were assessed in vitro for application in subsequent in vivo experiments. In the in vivo experiment, gene electrotransfer of pIL-2 and pIL-12 using the EP1 protocol was performed in B16.F10 murine melanoma. Combined treatment of tumours using pIL2 and pIL12 induced significant tumour growth delay and 71% complete tumour regression. Furthermore, in tumours coexpressing IL-2 and IL-12, increased accumulation of dendritic cells and M1 macrophages was obtained along with the activation of proinflammatory signals, resulting in CD4 + and CD8 + T-lymphocyte recruitment and immune memory development in the mice. In conclusion, we demonstrated high antitumour efficacy of combined IL-2 and IL-12 gene electrotransfer protocols in low-immunogenicity murine B16.F10 melanoma.

Histochemical characterization of the mucins of the alimentary tract of the grass snake, Natrix natrix (Colubridae).

Characterization of mucins in the alimentary tract of the grass snake, Natrix natrix was performed by histochemical (PAS, Alcian Blue, pH 2.5 and pH 1.0, sialidase-Alcian Blue, pH 2.5, HID-AB pH 2.5) and lectin-histochemical (WGA, SWGA, PNA, sialidase-PNA, SBA, sialidase-SBA, DBA, sialidase-DBA, ConA, BSI-B4, AAA, UEA-1, LTA) techniques. Oesophageal lining epithelium consisted of ciliated and goblet cells, with no pluricellular glands. Mannosylated sialosulfomucins were observed. Fundic mucosa of stomach presented surface cells producing sialomucins with terminal sialic acid linked to galactose. In gastric glands neck and oxynticopeptic cells were found. Neck cells had sialomucins with mannose, N-acetylglucosamine, galactose, N-acetylgalactosamine and fucose-α-(1,2)-linked residues. Cytoplasm of oxynticopeptic cells showed N-acetylgalactosamine and fucose residues. Secretion of surface cells in pyloric mucosa was similar to that of fundic ones, differing in having fucose. Goblet cells in the small intestine of N. natrix produced sulfo- and sialomucins, with sialic acid linked to galactose and N-acetylgalactosamine residues. Mucins also presented residues of mannose. Goblet cells in the large intestine presented sulfomucins only, with terminal N-acetylgalactosamine, galactose and N-acetylglucosamine. The glycosylation patterns found are probably related to protection against injuries, gastric juice and microorganisms, both pathogenic and decomposers, as well as to dietary adaptations.

[Placental insufficiency and intrauterine growth retardation]. / Insufficienza placentare e ritardo di crescita fetale.

AIM: Placental insufficiency is a pathological condition consisting of a placental functional deficit with multifactorial etiology; it can cause maternal complications such as edema, proteinuria, hypertension, etc. Our study aims to establish if placenta analysis after birth can lead to the identification of basic morphological alterations which can be easily documented and useful for the diagnosis of feto-neonatal pathologies. METHODS: We examined 60 pregnant women (45 primipara, 15 multipara). They were hospitalized in the period from March 1998 to March 2004 in different pregnancy periods because of fetus growth delay. After birth, a careful examination of the placenta has been carried out soon after spontaneous birth (weight, thickness, possible morphological alterations which could be macroscopically observed) and it was followed by an anatomo-pathological examination carried out at the Anatomopathology Unit of Ospedali Riuniti in Foggia. RESULTS: Macroscopic examination, in the group of women at the first pregnancy, showed that there were no morphological alterations in the placenta in all the cases, but the weight was normal (400- 500 g) in 8 cases and it was less than 400 g in the other 37 cases. In the group of multipara, placenta did not shown morphological alterations in all the cases, but the weight was normal in only 3 cases and it was lower in the remaining 12 cases. The placental microscopic examination pointed out: infarct focus in 34 cases (60%), 12 (35%) of which were recent and 22 (65%) were old infarct focuses; immaturity of chorionic villus in 15 (30%) of the examined placentas; increase in the cytotrophoblast mitotic index in 49 cases (90%) and placenta with normal histological appearance in 11 cases (10%). CONCLUSION: Placental examination, both with macroscopic and microscopic techniques, can lead to the identification of basic morphological alterations which can be easily documented and useful for the diagnosis of feto-neonatal pathologies. A good placental functionality influences both fetal life and postnatal life. Thus, a greater attention to the placental examination in obstetric practice is suggested, especially for maternal and fetal pathologies.

Mucin histochemistry of the digestive tract of the red-legged frog Rana aurora aurora.

Mucous cells from the digestive tract of the red-legged frog, Rana aurora aurora, were examined by standard histochemical methods and by lectin histochemistry. Two different goblet cell types were found in the oesophageal epithelium. Type I cells produced acidic glycoproteins with beta1,4GlcNAc oligomers, Gal beta1,3GalNAc sequences, sulphated esters on internal residues and abundant non-O-acylated terminal sialic acid bound to penultimate GalNAc. These cells also reacted with Con-A after periodate oxidation-borohydride reduction (PCS). Type II goblet cells mainly differed from type I cells in their negative reaction with PCS. Oesophageal glands consisted of mucous and serous cells. Mucous cells produced neutral stable class III mucosubstances with GalNAc, beta1,4GlcNAc and Gal beta1,3GalNAc residues. Gastric surface cells produced sulpho-sialoglycoproteins with Gal beta1,3GalNAc residues and Gal beta1,3GalNAc-sialic acid as terminal sequences. These cells did not contain stable class III mucosubstances. The mucus produced by foveolar cells was similar in composition but did not contain sulphated groups and was rich in GalNAc residues. The fundic glands consisted of mucous neck cells, endocrine cells and oxyntic cells. The neck cells produced neutral mucins containing D-mannose and/or D-glucose, beta1,4GlcNAc oligomers and Gal beta1,3GalNAc terminal dimers and were PCS-positive. Pyloric glands were of the mucus-secreting type, which produced glycoproteins with the same basic features as those produced in fundic neck cells. A single type of intestinal goblet cells produced acidic glycoproteins rich in beta1,4GlcNAc oligomers, sulphated esters on oligosaccharide chains and terminal O-acylated sialic acid bound to penultimate Gal beta1,3GalNAc. The different carbohydrate structures observed along the digestive tract of the frog may reflect specific functions of the mucus.

Fine structure of the oxynticopeptic cells in the gastric glands of the ruin lizard, Podarcis sicula campestris De Betta, 1857.

The results of an ultrastructural investigation of the gastric glands of the ruin lizard are reported. In this reptile the stomach can be divided into a larger fundus and a smaller pars pilorica. Fundic glands are characterized by three main kinds of cells: mucous, endocrine, and oxynticopeptic; the latter were not observed in the pyloric glands. The morphological features of the oxynticopeptic cells change from the proximal to the distal region of the fundic mucosa. In the proximal region, numerous electron-dense secretory granules, a well-developed granular endoplasmic reticulum, an evident Golgi complex, and a reduced system of smooth-surfaced vesicles and tubules in the apical cytoplasm characterize these cells. In the distal fundic region, oxynticopeptic cells possessed numerous mitochondria and a well-developed smooth-surfaced endoplasmic reticulum, but secretory granules were rare. These data suggest the existence of a gradient in the production of proteolytic enzymes, and perhaps also of hydrochloric acid, along the oral-aboral axis of the stomach. The results are discussed with regard to the evolution of the gastric glands and of the digestive mechanism in vertebrates.

Morphological and histochemical variations of mucous and oxynticopeptic cells in the stomach of the seps, Chalcides chalcides.

Mucous and oxynticopeptic cells in the gastric mucosa of the seps, Chalcides chalcides (Linnaeus, 1758) were examined by standard histochemical staining methods and by lectin histochemistry. The epithelial mucous cells lining the surface of the stomach and the mucous cells of the fundic glands elaborated mainly neutral glycoproteins with beta(1,4)GlcNAc oligomers, GalNAc glycosidic residues and Gal beta1,3GalNAc terminal sequences. The mucous cells of the fundic glands were stained specifically with the Paradoxical Con A method. The mucosecreting cells of the pyloric glands produced neutral glycoproteins, with beta(1,4)GlcNAc oligomers, GalNAc residues and Gal beta1,3GalNAc terminal sequences. Terminal L-fucose bound to the penultimate GlcNAc residues, and/or difucosylated oligosaccharides were also present. The pyloric glands did not stain with the Paradoxical Con A procedure. The morphology of the oxynticopeptic cells changes from the oral to the aboral region of the fundic mucosa. In the oral fundic tract the oxynticopeptic cells showed cytoplasm filled with zymogen granules, while in the aboral fundic region these cells contained few zymogen granules and showed cytoplasm full of empty vesicles, typical of the acid secreting cells. A secretion gradient of proteolytic enzymes and hydrochloric acid along the fundic mucosa of the seps can be hypothesised.