Massimo Masetti

Vitellin degradation in developing embryos of the stick insect Carausius morosus

Abstract Two vitellins of the stick insect Carausius morosus are utilized progressively during embryonic development. The relative titre of each vitellin was determined as a function of the developmental time using rocket immunoelectrophoresis with the specific anti-vitellin serum. The vitellins differ in the rate of utilization, one declining faster than the other. The pattern of vitellin in embryos differing in developmental time was demonstrated by polyacrylamide gel electrophoresis and subjected to immunoblotting using an anti-vitellin serum. Polypeptides E20, E9 and E5 that appeared de-novo during embryonic development were identified as fragments of the vitellins originally accumulated in newly laid eggs. Embryos differing in developmental stages were exposed to [35S]-methionine in vitro. Polypeptides labelled in vitro differed in molecular weight from those reactive to the anti-vitellin serum. Based on the above observations, it is concluded that vitellin degradation in C. morosus entails a stepwise degradation of vitellin polypeptides leading to the appearance of several derivatives of smaller molecular masses.

Immunosuppression of TH2 responses in Trichinella spiralis infection by Helicobacter pylori neutrophil-activating protein

BACKGROUND The Helicobacter pylori neutrophil-activating protein (HP-NAP) is able to induce IL-12 expression by cells of innate immunity and to shift to T(H)1 human allergen-specific T(H)2 cells in vitro. OBJECTIVE We performed an in vivo investigation of the ability of HP-NAP to downmodulate the T(H)2 response induced in mice by Trichinella spiralis infection. METHODS Groups of T spiralis-infected BALB/c mice received intraperitoneal PBS/rat IgG2b (control animals) or 10 microg of HP-NAP with or without anti-Toll-like receptor 2 antibody on days 10 and 28 after infection. Blood eosinophils, total and T spiralis-specific IgE levels, and cytokine levels were measured in the plasma up to day 42, when splenocytes were cultured for cytokine production. RESULTS Although control animals showed significant eosinophilia and increase of total and T spiralis-specific IgE, IL-4, and IL-5 levels from days 10 to 14, HP-NAP-treated animals showed less eosinophilia and total and excretory/secretory antigens of T spiralis-specific IgE in the blood. HP-NAP-treated animals also had higher IL-12 and IFN-gamma plasma levels and lower IL-4 and IL-5 levels. The addition of anti-Toll-like receptor 2 antibody abrogated the anti-T(H)2/pro-T(H)1 activity of HP-NAP. CONCLUSION This study provides evidence that HP-NAP enhances endogenous IL-12 and IFN-gamma response and exerts a powerful anti-T(H)2 activity in vivo, targeting both IL-5-induced eosinophilia and IL-4-mediated hyper-IgE responses induced by parasitic infection.

Yolk granules are differentially acidified during embryo development in the stick insect Carausius morosus

Abstract. Newly laid eggs of stick insects comprise a unique fluid ooplasm that is gradually partitioned into a number of yolk granules by invasion of secondary vitellophages. This study aimed at establishing how yolk granules become acidified in the course of embryonic development. Data show that acidified yolk granules are rather scarce and randomly distributed in vitellophages of early embryos, while they tend to increase gradually in number as development proceeds to completion. Yolk granule acidification is progressively more inhibited in the presence of increasing concentrations of chloroquine, monensin and bafilomycin. A pro-protease was identified cytochemically and by immunoblotting in yolk extracts of progressively more advanced embryos. A specific monoclonal antibody raised against this pro-protease helped to demonstrate that it is gradually processed to yield a lower molecular weight polypeptide as development proceeds to completion. This latter polypeptide was identified as a protease using electrophoresis in polyacrylamide gels containing yolk extracts. Simultaneous administration of a fluorescent substrate for cysteine protease and an acidotropic probe produced superimposable labelling patterns, suggesting that only acidified yolk granules possess a proteolytic activity. On the other hand, yolk granules probed simultaneously for acidification and latent pro-protease yielded labelling patterns partially superimposed. Pro-protease labelling is gradually lost as yolk granules are progressively more acidified during development. Distinct labelling patterns were also obtained in vitellophages processed for the simultaneous detection of pro-protease and protease, suggesting that the two activities are expressed by different yolk granule populations, and that one is gradually converted into the other as time goes by.

Larval salivary gland proteins of the sheep nasal bot fly, (Oestrus ovis L.), are major immunogens in infested sheep

Tissue extracts from larval instars of the sheep nasal bot, Oestrus ovis, were resolved by gel electrophoresis under both native and denaturing conditions. Polypeptides resolved under these conditions were tested by immunoblotting against sera of infested sheep. Of all tissues examined in this study, salivary glands proved to be major immunogens in infested sheep. Salivary gland polypeptides were also detected in the washing solution as larval secretory products (LSP). To a minor extent, a few polypeptides from the larval cuticle were also found to be immunogenic, but they did not contribute to LSP. These results were further corroborated by nasal infestation of rabbits that also developed specific antibodies against larval salivary gland polypeptides from Oestrus ovis.

The use of mercury against pediculosis in the Renaissance: the case of Ferdinand II of Aragon, King of Naples, 1467-96.

The hair samples of Ferdinand II of Aragon (1467–1496), King of Naples, whose mummy is preserved in the Basilica of San Domenico Maggiore in Naples, showed a high content of mercury, with a value of 827ppm. Furthermore, examination using a stereomicroscope and a scanning electron microscope (SEM) of head and pubic hairs of Ferdinand II, revealed a lice infestation. The reasons for the massive presence of the mercury in the kings hair are discussed and contemporary literature regarding the use of this metal in medical therapies and in cosmetic practices is analysed. As a result, the high value of mercury in the hair of Ferdinand II can be attributed to antipediculosis therapy, applied as a topic medicament. This case represents an important finding for the history of medicine, because demonstrates that in the Renaissance mercury was applied locally not only to treat syphilis, as well attested by direct and indirect sources, but also to prevent or eliminate lice infestation.

Royal pediculosis in Renaissance Italy: lice in the mummy of the King of Naples Ferdinand II of Aragon (1467-1496)

Pediculosis seems to have afflicted humans since the most ancient times and lice have been found in several ancient human remains. Examination of the head hair and pubic hair of the artificial mummy of Ferdinand II of Aragon (1467-1496), King of Naples, revealed a double infestation with two different species of lice, Pediculus capitis, the head louse, and Pthirus pubis, the pubic louse. The hair samples were also positive for the presence of mercury, probably applied as an anti-pediculosis therapy. This is the first time that these parasites have been found in the hair of a king, demonstrating that even members of the wealthy classes in the Renaissance were subject to louse infestation.

Evaluation of inflammatory responses against muscle larvae of different Trichinella species by an image analysis system

The aim of this study was to evaluate the inflammatory response in the muscle tissue against Trichinella larvae of encapsulated (T. spiralis, T. britovi) and non-encapsulated (T. pseudospiralis) species. The inflammatory response was estimated in histological sections of muscle tissues from Trichinella-infected CD1 mice by a newly developed method. Nuclei were stained with one fluorescent probe, which binds nucleic acids with high affinity, and fluorescence was analysed by a software program. Evaluation of the relative fluorescence units was performed in both peri-capsular (close to the nurse cell-parasite complex) and extra-capsular (where the parasite was not visible) areas. The increase in the number of nuclei in the muscle tissues of Trichinella-infected mice was considered an inflammation marker, since uninfected muscles show low nucleus density. In order to evaluate differences in the nitrosylation pattern between encapsulated (T. spiralis, T. britovi) and non-encapsulated (T. pseudospiralis, Trichinella papuae, Trichinella zimbabwensis) species, L(1) larvae were tested by immunoblotting with an anti-nitrotyrosine polyclonal antibody. Inflammation induced by T. spiralis larvae in muscle tissues is statistically higher than that elicited by the other species, both in peri- and extra-capsular areas. Nitrosylation occurs at a higher level in encapsulated than in non-encapsulated species. The method developed in this work allows demonstration of differences in the host inflammatory response against encapsulated and non-encapsulated Trichinella species.

Establishment and characterization of 4 new human pancreatic cancer cell lines: evidences of different tumor phenotypes.

Objectives: Pancreatic cancer still remains a challenge for its biological complexity and lack of effective therapeutic strategies. Establishing new pancreatic cancer cell lines is therefore of paramount importance to clarify its biology. Methods: We established and characterized 4 new pancreatic cancer cell lines (PP78, PP109, PP117, and PP161) according to their genetic (K-Ras, TP53, CDKN2A, and MADH4; DNA fingerprinting; karyotype), cytostructural (cytokeratins 7, 8, 18, and 19 vimentin, and ezrin), and functional profiles (doubling time; migration assay). Results: K-Ras, TP53, and CDKN2A gene alterations were detected in all 4 of them. Each cell line had a unique DNA profile revealed by DNA fingerprinting. A complex karyotype with numerous structural and numeric chromosomal abnormalities was present in each cell line. All 4 cell lines showed positivity for cytokeratins 7, 8, and 18. All but PP78 expressed cytokeratin 19, whereas vimentin was expressed only in PP117 and PP78 cells. A different ezrin cellular distribution was noticed in PP78 and PP117, being mostly located at membrane ruffles. This peculiar distribution was associated with the strongest migratory capability. Conclusions: Our results seem to confirm the pancreatic ductal adenocarcinoma heterogeneity; in fact, the same genetic abnormalities (K-Ras, TP53, and CDKN2A) may have different effects on tumor biology depending on cellular differentiation.

Mono- and polyclonal antibodies as probes to study vitellin processing in embryos of the stick insect Carausius morosus

During embryonic development, insect vitellins (Vt) are degraded by limited proteolysis to yield a number of lower-molecular weight polypeptides. The aim of the present study was to identify these polypeptides in the embryo and to verify how they relate to Vt polypeptides deposited in the oocyte during vitellogenesis. To this end a panel of poly- and monoclonal antibodies (Pab, Mab) was raised against Vt polypeptides and employed by immunoelectrophoresis and immunoblotting on embryos belonging to different developmental stages. Through this approach three major staining patterns were observed. First, Mab 4 reacts with both polypeptides B1 and E20, suggesting that polypeptide B1 is gradually trimmed to yield polypeptide E20 in late embryos. Second, Mab 12 is specific for polypeptide A3 which is retained unchanged throughout embryogenesis. Third, Pab anti-A2 and Mab 13 show that polypeptide A2 is processed to yield polypeptide E9 through limited proteolysis. In conclusion, the staining patterns reported in this study show that Vt polypeptides in developing embryos of the stick insect Carausius morosus undergo at least two major processing events concerning polypeptides B1 and A2.

Vitellogenesis in the allatectomized stick insect Carausius morosus (br.) (Phasmatodea: Lonchodinae)☆

Abstract Effects of allatectomy on vitellogenesis in adult Carausius morosus Br. were examined using rocket immunoelectrophoresis, polyacrylamide gel electrophoresis, fluorographic identification and quantitation by liquid scintillation of in vivo35S-methionine-labeled proteins, and light microscope autoradiography. In normal adults and in adults allatectomized as last instar nymphs, Coomassie Blue-stained vitellogenin (Vg) was first detectable in the hemolymph between 3 and 5 days after adult emergence. Allatectomy of 11–18-day-old adults produced no detectable effects on subsequent Vg synthesis, secretion or uptake, but ovarian follicles in adults that had been allatectomized as nymphs were less efficient at taking up Vg than were those in sham-operated animals. Compared to sham-operated controls, adults allatectomized as nymphs displayed an increased rate of accumulation of newly synthesized Vg in the hemolymph, a decreased rate of accumulation of vitellin in terminal follicles, a decrease in the size of yolk spheres containing newly synthesized vitellogenic protein in the peripheral ooplasm, and alterations in the size distribution of terminal follicles. Thus, post-induction vitellogenesis and primary induction of Vg synthesis in C. morosus do not require active corpora allata (CA), but a normal rate of Vg uptake may be JH-facilitated.