Publications by authors named "Gennaro D"

27 Publications

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The invasive mosquito Aedes japonicus japonicus is spreading in northeastern Italy.

Parasit Vectors 2019 Mar 26;12(1):120. Epub 2019 Mar 26.

Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Italy.

Background: The invasive mosquito species, Aedes japonicus japonicus, was detected in northeastern Italy for the first time in 2015, at the border with Austria. After this finding, a more intensive monitoring was carried out to assess its distribution and to collect biological data. Herein, we report the results of four years (2015-2018) of activity.

Methods: The presence of Ae. j. japonicus was checked in all possible breeding sites through collections of larvae. The monitoring started from the site of the first detection at the Austrian border and then was extended in all directions. The mosquitoes were identified morphologically and molecularly.

Results: Aedes j. japonicus was found in 58 out of 73 municipalities monitored (79.5%). In total (2015-2018), 238 sampling sites were monitored and 90 were positive for presence of Ae. j. japonicus larvae (37.8%). The mosquito was collected mainly in artificial containers located in small villages and in rural areas. Cohabitation with other mosquito species was observed in 55.6% of the samplings.

Conclusions: Aedes j. japonicus is well established in Italy and in only four years has colonised two Italian Regions, displaying rapid spreading throughout hilly and mountainous areas. Colonization towards the south seems limited by climatic conditions and the occurrence of a large population of the larval competitor, Ae. albopictus. The further spread of Ae. j. japonicus has the potential to pose new threats of zoonotic agents (i.e. Dirofilaria spp. and West Nile virus) within areas at altitudes previously considered at negligible risk in Italy.
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http://dx.doi.org/10.1186/s13071-019-3387-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6434805PMC
March 2019

Pro-Degradant Activity of Naturally Occurring Compounds on Polyethylene in Accelerate Weathering Conditions.

Materials (Basel) 2019 Jan 8;12(1). Epub 2019 Jan 8.

Dipartimento di Ingegneria Civile, Ambientale, Aerospaziale, dei Materiali, Università di Palermo, Viale delle Scienze, Ed. 6, 90128 Palermo, Italy.

In this work, naturally occurring compounds, such as Vitamin E (VE) and Ferulic Acid (FA), at high concentrations, have been considered as pro-degradant agents for Low Density Polyethylene (PE). However, all obtained results using the naturally occurring molecules as pro-oxidant agents for PE have been compared with the results achieved using a classical pro-oxidant agent, such as calcium stearate (Ca stearate) and with neat PE. The preliminary characterization, through rheological, mechanical and thermal analysis, of the PE-based systems highlights that the used naturally occurring molecules are able to exert a slight plasticizing action on PE and subsequently the PE rigidity and crystallinity slightly decrease, while the ductility increases. To assess the pro-degradant activity of the considered naturally occurring compounds, thin films of neat PE and PE-based systems containing 2 and 3 wt.% Ca stearate, VE and FA have been produced and subjected to accelerated weathering upon UVB light exposure. All obtained results point out that the VE and FA, at these high concentrations, exert a clear pro-oxidant activity in PE and this pro-oxidant activity is very similar to that exerted by Ca stearate. Moreover, the VE and FA at high concentrations can be considered as suitable eco-friendly pro-degradant additives for PE, also in order to control the polyolefin degradation times.
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http://dx.doi.org/10.3390/ma12010195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6337242PMC
January 2019

Lemierre's syndrome: One rare disease-Two case studies.

J Clin Pharm Ther 2019 Feb 28;44(1):122-124. Epub 2018 Nov 28.

Touro College of Pharmacy, New York, New York.

What Is Known And Objective: Lemierre's syndrome is often misdiagnosed as a common cold or viral infection. Fusobacterium necrophorum is the most common causative organism. The recommended treatment regimen is 6 weeks of a beta-lactam antibiotic along with metronidazole.

Case Description: We present two cases of Lemierre's syndrome with internal jugular vein thrombophlebitis and positive blood cultures for F. necrophorum. The first case was successfully treated with 6 weeks of a beta-lactam antibiotic and 4 weeks of metronidazole, while the second case was successfully treated with 4 weeks of a beta-lactam antibiotic and 2 weeks of metronidazole.

What Is New And Conclusion: Two cases of Lemierre's syndrome were treated successfully with only 2-4 weeks of metronidazole therapy. Shorter duration of metronidazole therapy should be explored in future studies.
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http://dx.doi.org/10.1111/jcpt.12774DOI Listing
February 2019

Combined magnetic resonance imaging and high resolution spectroscopy approaches to study the fertilization effects on metabolome, morphology and yeast community of wine grape berries, cultivar Nero di Troia.

Food Chem 2019 Feb 11;274:831-839. Epub 2018 Sep 11.

Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria - Research Centre for Agriculture and Environment (CREA-AA), Via della Navicella 2, 4-00184 Roma, Italy. Electronic address:

Effects of fertilization practices, mineral (M) and organo-mineral (OM), on molecular composition of Nero di Troia cultivar grape berries was studied using conventional chemical analysis, Magnetic Resonance Imaging (MRI) and H NMR spectroscopy on intact berries and extracts, respectively, and through analysis of yeast species developed on grape skins. Plants vegetative status did not differ between the two fertilization practices, whereas some grape juice chemical characteristics differed in fertilized grapes. MRI provided information on grape berries morphology through weighted images depending on spin-spin (T2) and spin-lattice (T1) relaxation times. T1 values were the highest in OM grape berries. H NMR metabolic profile, combined with chemometric analysis, evidenced significant differences for some metabolites (valine, leucine, isoleucine, proline, and malic acid). Furthermore, higher frequency of yeasts genus Starmella sp., isolated from OM grape berries contributed to reinforcing the found results on the physiological response of wine grape Nero di Troia to fertilization.
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http://dx.doi.org/10.1016/j.foodchem.2018.09.056DOI Listing
February 2019

Major phenolic and volatile compounds and their influence on sensorial aspects in stem-contact fermentation winemaking of Primitivo red wines.

J Food Sci Technol 2016 Aug 29;53(8):3329-3339. Epub 2016 Aug 29.

Consiglio per la Ricerca in Agricoltura e L'Analisi dell'Economia Agraria - Cantina Sperimentale di Barletta, Via Vittorio Veneto 26, 76121 Barletta, Italy.

In red winemaking de-stemming is crucial since the stems contain polymeric phenolic compounds responsible for the astringency of wine. Wine such as Primitivo has low phenolic constituents and tannins and stems affect aroma, taste body and olfactory characteristics. The aim of the study was to evaluate the effects of presence of stems during fermentation on polyphenolic, volatile compounds and sensory characteristics of wine. Primitivo grapes vinified in presence of different percentage of stems: 100 % de-stemmed (D100), 75 % de-stemmed (D75) and 50 % de-stemmed (D50). Results confirmed that the wines vinified in presence of stems were higher in tannins, flavans, to vanillin and proanthocyanidins, colour intensity with lower anthocyanins. The presence of stems during fermentation conferred more structure and flavour to wines. They facilitated must aeration thus promoting synthesis of higher alcohols and ethyl esters by yeast. In particular, a higher content of hexan-1-ol, hex-3-en-1-ol and 2-phenyl ethanol in D50 and D75 gave the wines that suggest green grass, herb and floral. Wine from D75 seemed to be better than D50 in terms of volatile compounds as well as fruity, floral and balsamic components preserved, without any unpleasant taste of long chain fatty acids found in D50.
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http://dx.doi.org/10.1007/s13197-016-2310-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5055897PMC
August 2016

Maceration with stems contact fermentation: effect on proanthocyanidins compounds and color in Primitivo red wines.

Food Chem 2015 Jun 17;177:382-9. Epub 2015 Jan 17.

Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Unità di Ricerca per l'Uva da Tavola e la Vitivinicoltura in Ambiente Mediterraneo, Cantina Sperimentale di Barletta, Via Vittorio Veneto 26, 76121 Barletta, Italy. Electronic address:

Three Primitivo (Vitis vinifera, cv.) red wines were microvinified by means of different winemaking technologies: no stem-contact fermentation destemming 100% of grapes (D100); stem-contact fermentation destemming 75%, 50% of grapes (D75-D50) respectively. The objectives of this work were to improve proanthocyanidins content in wine, to monitor the relationships between anthocyanins/tannins and to detect the effects on the polymerization state of polyphenols after 12 months storage of wines. D100 showed higher anthocyanins content but lower color intensity and phenolic compounds content with respect to the theses D75 and D50, the last two showing lower anthocyanins content due their partial adsorption by grape stems during the fermentation. D75 gave the best results in terms of anthocyanins/color intensity balance and showed a better wine tannin component with respect to D50. Moreover after 12 months storage D50 reached a more advanced and stable polymerization state of colored pigments than the other wines.
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http://dx.doi.org/10.1016/j.foodchem.2015.01.063DOI Listing
June 2015

Maceration with stems contact fermentation: effect on proanthocyanidins compounds and color in Primitivo red wines.

Food Chem 2015 Jun 17;177:382-9. Epub 2015 Jan 17.

Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Unità di Ricerca per l'Uva da Tavola e la Vitivinicoltura in Ambiente Mediterraneo, Cantina Sperimentale di Barletta, Via Vittorio Veneto 26, 76121 Barletta, Italy. Electronic address:

Three Primitivo (Vitis vinifera, cv.) red wines were microvinified by means of different winemaking technologies: no stem-contact fermentation destemming 100% of grapes (D100); stem-contact fermentation destemming 75%, 50% of grapes (D75-D50) respectively. The objectives of this work were to improve proanthocyanidins content in wine, to monitor the relationships between anthocyanins/tannins and to detect the effects on the polymerization state of polyphenols after 12 months storage of wines. D100 showed higher anthocyanins content but lower color intensity and phenolic compounds content with respect to the theses D75 and D50, the last two showing lower anthocyanins content due their partial adsorption by grape stems during the fermentation. D75 gave the best results in terms of anthocyanins/color intensity balance and showed a better wine tannin component with respect to D50. Moreover after 12 months storage D50 reached a more advanced and stable polymerization state of colored pigments than the other wines.
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http://dx.doi.org/10.1016/j.foodchem.2015.01.063DOI Listing
June 2015

Maceration with stems contact fermentation: effect on proanthocyanidins compounds and color in Primitivo red wines.

Food Chem 2015 Jun 17;177:382-9. Epub 2015 Jan 17.

Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Unità di Ricerca per l'Uva da Tavola e la Vitivinicoltura in Ambiente Mediterraneo, Cantina Sperimentale di Barletta, Via Vittorio Veneto 26, 76121 Barletta, Italy. Electronic address:

Three Primitivo (Vitis vinifera, cv.) red wines were microvinified by means of different winemaking technologies: no stem-contact fermentation destemming 100% of grapes (D100); stem-contact fermentation destemming 75%, 50% of grapes (D75-D50) respectively. The objectives of this work were to improve proanthocyanidins content in wine, to monitor the relationships between anthocyanins/tannins and to detect the effects on the polymerization state of polyphenols after 12 months storage of wines. D100 showed higher anthocyanins content but lower color intensity and phenolic compounds content with respect to the theses D75 and D50, the last two showing lower anthocyanins content due their partial adsorption by grape stems during the fermentation. D75 gave the best results in terms of anthocyanins/color intensity balance and showed a better wine tannin component with respect to D50. Moreover after 12 months storage D50 reached a more advanced and stable polymerization state of colored pigments than the other wines.
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http://dx.doi.org/10.1016/j.foodchem.2015.01.063DOI Listing
June 2015

Diarrhoea in irradiated patients: a prospective multicentre observational study.

Dig Liver Dis 2013 Nov 20;45(11):933-7. Epub 2013 Jun 20.

Radiotherapy Centre, "G. Martino" University Hospital, Messina, Italy. Electronic address:

Aims: To determine the incidence of cancer treatment-induced diarrhoea in patients submitted to irradiation.

Methods: Forty-five Italian radiation oncology departments took part in this prospective observational study and a total of 1020 patients were enrolled. The accrual lasted three consecutive weeks; evaluation was based on diary cards filled in daily by patients during radiotherapy and one week after cessation. Diary cards recorded both the onset and intensity of diarrhoea.

Results: A total of 1004 patients were eligible for this analysis. 147/1004 (14.6%) patients had diarrhoea. The median minimum number of daily events was 1 (range 1-7) with a median maximum events of 3 (range 1-23). 82/147 patients (56.2%) had a drug prescription for diarrhoea. In the evaluation of the onset of diarrhoea, in multivariate analysis, we found the following factors to be statistically significant predictors of an increased likelihood of diarrhoea: primitive tumour site, therapeutic purpose and field size.

Conclusions: Patients with abdominal-pelvic cancer, treated with curative purpose and using large field sizes are at high risk of cancer treatment-induced diarrhoea. Diarrhoea was also observed in patients treated at other sites. In this population group there is the need for more stringent monitoring during the delivery of radiation therapy.
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http://dx.doi.org/10.1016/j.dld.2013.04.012DOI Listing
November 2013

Skin toxicity from external beam radiation therapy in breast cancer patients: protective effects of Resveratrol, Lycopene, Vitamin C and anthocianin (Ixor®).

Radiat Oncol 2012 Jan 30;7:12. Epub 2012 Jan 30.

Dipartimento Diagnostica per Immagini e Radioterapia Seconda Università di Napoli-P.zza Luigi Miraglia-(80138) Napoli, Italy.

Introduction: This is an observational study and the aim is to evaluate the effect of dietary supplements based on Resveratrol, Lycopene, Vitamin C and Anthocyanins (Ixor®) in reducing skin toxicity due to external beam radiotherapy in patients affected by breast cancer.

Materials And Methods: 71 patients were enrolled and they were divided in two different groups: a control group (CG) of 41 patients treated with prophylactic topical therapy based on hyaluronic acid and topical steroid therapy in case of occurrence of radiodermatitis, and a Ixor-Group (IG) of 30 patients treated also with an oral therapy based on Resveratrol, Lycopene, Vitamin C and Anthocyanin (Ixor®) at a dose of 2 tablets/day, starting from 10 days before the radiation treatment until 10 days after the end of treatment. Skin toxicity has been related to PTV, to breast volume that received a radiation dose equal or lower than 107%, included between 107% and 110%, or greater than 110% of the prescribed dose. Moreover it's been studied the relationship between skin toxicity and the chemotherapy schedule used before treatment. We calculated in both groups the percentage of patients who had a skin toxicity of grade 2 or 3 (according to RTOG scale). Absolute risk reduction (ARR), relative risk (RR) and odds ratio (OR) have been calculated for each relationship.

Results: Control Group (CG) patients with a PTV > 500 ml presented skin toxicity G2 + G3 in 30% of cases, versus 25% of Ixor-Group (IG) [OR 0.77]. In patients with a PTV < 500 ml G2 + G3 toxicity was 0% in the IG compared to 18% in CG (OR 0.23). When Dmax was less than or equal to 107% of the prescribed dose skin toxicity was G2 + G3 in 12.5% in CG, versus 0% in IG (OR 0.73), instead when Dmax was included between 107 and 110% of the prescribed dose, G2 + G3 skin toxicity was 35% in CG and 21% in IG (OR 0.50). In patients undergoing chemotherapy with anthracyclines and taxanes, G2 + G3 toxicity was 27% in CG, against 20% in IG (OR 0.68).

Conclusions: The protective effect of Resveratrol, Lycopene, Vitamin C and Anthocyanin (Ixor®) is more detected in patients with PTV < 500 ml, when Dmax reaches values lower or equal to 107%, but not exceeding 110% of the prescribed dose, and in patients undergoing adjuvant chemotherapy with anthracyclines and taxanes.
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http://dx.doi.org/10.1186/1748-717X-7-12DOI Listing
January 2012

A prospective observational trial on emesis in radiotherapy: analysis of 1020 patients recruited in 45 Italian radiation oncology centres.

Radiother Oncol 2010 Jan 4;94(1):36-41. Epub 2009 Dec 4.

Radiotherapy Centre, S. Maria Hospital, Via T. di Joannuccio, 1, O5100 Terni, Italy.

Purpose: A prospective observational multicentre trial was carried out to assess the incidence, pattern, and prognostic factors of radiation-induced emesis (RIE), and to evaluate the use of antiemetic drugs in patients treated with radiotherapy or concomitant radio-chemotherapy. The application in clinical practice of the Multinational Association of Supportive Care in Cancer guidelines was also studied.

Materials And Methods: Forty-five Italian radiation oncology centres took part in this trial. The accrual lasted for 3 consecutive weeks and only patients starting radiotherapy or concomitant radio-chemotherapy in this period were enrolled. Evaluation was based on diary card filled in daily by patients during treatment and one week after stopping it. Diary card recorded the intensity of nausea/vomiting and prophylactic/symptomatic antiemetic drug prescriptions.

Results: A total of 1020 patients entered into the trial, and 1004 were evaluable. Vomiting and nausea occurred in 11.0% and 27.1% of patients, respectively, and 27.9% patients had both vomiting and nausea. In multifactorial analysis, the only statistically significant patient-related risk factors were concomitant chemotherapy and previous experience of vomiting induced by chemotherapy. Moreover, two radiotherapy-related factors were significant risk factors for RIE, the irradiated site (upper abdomen) and field size (>400 cm(2)). An antiemetic drug was given only to a minority (17%) of patients receiving RT, and the prescriptions were prophylactic in 12.4% and symptomatic in 4.6%. Different compounds and a wide range of doses and schedules were used.

Conclusions: These data were similar to those registered in our previous observational trial, and the radiation oncologists' attitude in underestimating RIE and under prescribing antiemetics was confirmed.
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http://dx.doi.org/10.1016/j.radonc.2009.11.001DOI Listing
January 2010

Prognostic value of biophysical profile score in post-date pregnancy.

Clin Exp Obstet Gynecol 1995 ;22(4):330-8

2nd Department of Obstetrics and Gynecology, University of Rome, La Sapienza.

Objectives: This study was undertaken to assess the ability of each individual biophysical profile score variable and combination of variables, to predict fetal distress or imminent labor in the post data pregnancy.

Materials And Methods: From June 1992 to August 1993, Biophysical Profile Scoring (BPS) was performed on 182 pregnant women. Thirty one patients delivered between 42 and 43 weeks of gestation, while the other 151 pregnant women delivered between 38 and 41+6 days. Monitoring procedures were based on the evaluation of fetal heart rates by Non Stress Test and four ultrasound parameters: Fetal Tone; Gross Body Movements; Fetal breathing movements; Evaluation of amniotic fluid volume.

Results: In this analysis the BPS showed a high specificity (82.7%) with a negative predictive value of 100%. The mean value of the last BPS among the 151 term patients was significantly higher if compared with the 31 post term pregnant women (6.73 and 6.12 respectively with p < 0.05). The predictive value of Fetal Breathing Movements (FBM) for the onset of imminent labor was confirmed.

Conclusion: BPS, as measured in this study, has proved to be a very accurate method of determination of fetal well-being. Although these results will be further verified by other studies, BPS should come into general use to help reach the correct diagnosis and treatment of post date pregnancy.
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September 1996

[Cervicofacial neuroendocrine Merkel cell carcinoma: radiotherapy].

Acta Otorhinolaryngol Ital 1994 Nov-Dec;14(6):651-7

Divisione di Otorinolaringoiatria, Ospedali Riuniti, Salerno.

The Authors report a case of Merkel cell carcinoma of the cervicofacial district, particular due to its clinical and evolutive characteristics. The Merkel cell carcinoma was first identified by Toker in 1972 and is also known as a primary neuroendocrine the or trabecular carcinoma of the skin. The Merkel cell carcinoma is a rare aggressive skin cancer most frequently localized in the cervicofacial district. Although in earlier reports on the clinical history of these tumors primary emphasis was placed on their local aggressive behaviour, it has recently become evident that these neoplasms have a marked propensity for early dissemination. Distant metastases are often responsible for disease-related mortality. Merkel cell carcinoma have classically been managed with surgery alone or surgery followed by radiation therapy. Treatment of Merkel cell carcinoma with radiation therapy alone has usually been reserved for advanced unresectable tumors.
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June 1995

[Comparison of echo-Doppler and splenoportography in the hemodynamic evaluation of the portal system].

Minerva Chir 1992 Dec;47(23-24):1793-8

Dipartimento di Discipline Chirurgiche, Università degli Studi di Palermo.

The Authors discuss the instrumental diagnosis of portal hypertension, comparing echo-Doppler and splenoportography by celiac artery catheterization, performed on 42 patients with cirrhosis. From the data obtained, there may be observed a clear correlation between angiographic and echographic criteria, correlated to the degree of illness. In particular echo-Doppler whether it possible, in a non-invasive way, to control the evolution of progressive haemodynamic resistance, and hepatic and splenic parenchyma, leaving to splenoportography the role to depict anatomic characteristics of portal circulation for a correct surgical program.
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December 1992

[Diagnostic "gold standard" in cerebrovascular insufficiency. Comparison of angiographic and ultrasonic imaging].

Minerva Chir 1992 Aug;47(15-16):1241-8

Dipartimento di Discipline Chirurgiche ed Anatomiche, Università degli Studi di Palermo.

The Authors consider the complex diagnostic problems of cerebrovascular disorders, with particular concern to angiography and echography. After a complete analysis of different aspects of angiographic and echographic lesions, considering their experience, they identify chances and limits of the two methods, they draw the conclusion that each of the two techniques has a proper role, and only a correct correlation between them can depict correctly the pathologic lesions.
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August 1992

Quantitative immunocytochemical staining for recombinant tissue-type plasminogen activator in transfected Chinese hamster ovary cells.

Proc Soc Exp Biol Med 1991 Oct;198(1):591-8

Department of Experimental Pathology, SmithKline Beecham Pharmaceuticals, Philadelphia, Pennsylvania 19406.

Tissue-type plasminogen activator (tPA) is a serine protease which cleaves plasminogen to its active form, plasmin. tPA plays a physiologic role in hemostasis, wound healing, and embryogenesis. Therapeutically, recombinant human tPA is used as a thrombolytic in myocardial infarction. Although production of therapeutic quantities of tPA in Chinese hamster ovary (CHO) cells transfected with the human gene for tPA is practical, production costs remain high. One important factor which determines the ultimate cost of tPA (or any other recombinant protein expressed in mammalian cells) is its production level on a per cell basis. We have used postembedding immunocytochemical staining with colloidal gold to study the subcellular localization of tPA in CHO cells expressing recombinant tPA (rCHO) in an effort to understand the factor(s) which might limit secretion. Staining for tPA was evaluated visually and by morphometric analysis and was specific and reproducible. Serially passaged rCHO showed no significant change in staining density over 31 serial passages. Staining density was greatest over dilated cisternae of the rough endoplasmic reticulum and nuclear envelope. Golgi stacks and large acid phosphatase-positive vacuoles (probably lysosomes) were also heavily stained. Staining of lysosomal vacuoles suggested that rCHO might be degrading nascent tPA. Incubation of rCHO with 125I-tPA showed that the cells were not internalizing tPA from the media. These results suggest that rCHO fail to secrete a portion of the tPA they synthesize and that it is degraded in lysosomes. This observation may have important implications on the choice of expression systems for efficient production of large quantities of recombinant proteins.
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http://dx.doi.org/10.3181/00379727-198-43294DOI Listing
October 1991

A new cytochemical method for ultrastructural detection of liposomes in tissues in vivo.

J Histochem Cytochem 1989 Jun;37(6):843-51

Department of Experimental Pathology, Smith Kline & French Laboratories, Philadelphia, Pennsylvania 19101.

Multilamellar vesicles (MLVs) have been used as drug carriers to increase efficacy or decrease toxicity of a variety of therapeutic agents, including antineoplastics, antibiotics, and immunomodulators. Although analysis of the disposition of encapsulated materials is relatively simple using radiolabels or single enzymes, determining the cellular and subcellular disposition of intact MLVs, i.e., those that still retain their encapsulated materials, is much less straightforward. We have developed a technique that allows demonstration of the uptake of intact MLVs by Kupffer cells. The method is based on co-localization of paired enzymes, glucose oxidase (GO), and horseradish peroxidase (HRP). The rationale for the localization is that H2O2 generated from glucose and oxygen by GO acts as the substrate for the HRP-mediated oxidative polymerization of diaminobenzidine. Therefore, only sites of co-localization of GO and HRP should stain. Mice were injected IV with phosphatidyl choline MLVs encapsulating HRP and GO. Encapsulated enzymes were separated from non-encapsulated by passing the MLVs over a Sepharose 2B column. Control mice were injected with equivalent amounts of free GO. Mice were sacrificed 30 min after injection and liver tissue was fixed in 3% cacodylate-buffered glutaraldehyde for at least 18 hr. Tissues were washed in buffer, then stained in medium containing glucose, diaminobenzidine HCl, and dimethylsulfoxide in 0.1 M cacodylate buffer. In animals injected with MLV-encapsulated GO and HRP, vacuoles in Kupffer cells and some endothelial cells contained electron-dense reaction product. No other cell type, including polymorphonuclear leukocytes, was stained. In control animals no staining was seen. Our results indicate that encapsulation of paired enzymes may be a feasible method to demonstrate the cellular and subcellular disposition of intact liposomes.
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http://dx.doi.org/10.1177/37.6.2723402DOI Listing
June 1989

Cytochemical demonstration of constitutive H2O2 production by macrophages in synovial tissue from rats with adjuvant arthritis.

Am J Pathol 1988 Jan;130(1):120-5

Smith Kline & French Laboratories, Philadelphia, Pennsylvania.

Generation of toxic oxygen metabolites by inflammatory cells is considered to be one of the mechanisms by which inflammation produces tissue injury. This concept is based on in vitro studies of purified leukocyte populations because it has not been possible to assess production of these metabolites in tissues. In order to determine whether or not inflammatory cells in tissue generate H2O2, the authors modified an earlier cytochemical method for the localization of H2O2. The incubation medium consists of 0.5 mM CeCl3 in a Hepes-buffered balanced salt solution with Cl- as the only anion. Synovial tissue from the knees of normal and 16-day adjuvant arthritic rats was incubated in this medium for 30 minutes and then fixed and processed for electron microscopy. No H2O2 reaction product was visible in normal synovium. In contrast, patchy deposits of H2O2 reaction product were seen adjacent to a subpopulation of synovial lining macrophages in synovium from inflamed knee joints. These data show that rat synovial macrophages are capable of generating H2O2 when appropriately stimulated and that such a stimulus is present in adjuvant arthritis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1880542PMC
January 1988

Endocytosis and de novo expression of major histocompatibility complex encoded class I molecules: kinetic and ultrastructural studies.

Eur J Cell Biol 1987 Dec;45(1):126-36

Department of Cell Biology, Smith Kline and French Laboratories, Philadelphia, PA.

The endocytic pathway and expression of the major histocompatibility complex encoded class I molecule H-2Kk was investigated in murine fibroblasts. Internalization of H-2K molecules did not occur constitutively. Endocytosis of the molecules was induced by addition of multivalent ligands such as rabbit anti-mouse immunoglobulin serum or protein A-bearing liposomes to cells pretreated with anti-H-2Kk antibodies. The complete removal of H-2K molecules took about 5 h at 37 degrees C and was not inhibited by the lysosomotropic agent NH4Cl or the protein synthesis inhibitor cycloheximide. When targeted liposomes that contained carboxyfluorescein at a self-quenched concentration were directed against H-2K molecules, the cells became highly fluorescent after 30 min: a consequence of carboxyfluorescein release from the liposomes. This process was inhibited by NH4Cl but not by cycloheximide, suggesting internalization of H-2K molecules into acidic intracellular compartments. The endocytic pathway of liposomes directed against H-2K molecules and the subcellular compartments involved in this process were investigated with targeted liposomes containing horseradish peroxidase. By electron microscopy, the endocytic process was shown to start very rapidly (1-2 min) and involved uncoated cell surface invaginations. The cytoplasmic uncoated vesicles fused together into larger vacuoles containing concentrated liposomes and by 1 h, liposomes began to be destroyed in lysosomal compartments. Within 4 h, 90% of liposomes were lysed inside the cell. The fate of radiolabeled anti-H-2K antibody was also investigated. Degradation of the antibody occurred only when cross-linked with a second layer of antibody, beginning after 2 h and becoming more pronounced after 20 h of incubation. The original cell surface abundance of H-2K molecules was reestablished after 5 to 7 h. During this time neither NH4Cl nor cycloheximide had any effect on the cell surface expression of the molecule. However, after a second cycle of internalization, cells incubated with cycloheximide no longer expressed these molecules. These results suggested that H-2K molecules were not recycled back to the surface after internalization but were degraded in lysosomal compartments together with their ligand. Preexisting molecules, already present in intracellular pools, were expressed to replace them. By immunoprecipitation of metabolically labeled intracellular and surface H-2K molecules, we observed an intracellular pool of H-2K of about 70 to 80% of the total cellular H-2K.
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December 1987

Major histocompatibility complex class I molecules internalized via coated pits in T lymphocytes.

Nature 1987 Aug 20-26;328(6132):724-6

Endocytosis of the major histocompatibility complex (MHC)-encoded class I and class II molecules has been the subject of recent investigations. Class I molecules, which are key elements in T cell-mediated cytotoxicity, are differentially endocytosed by different cell types. Fibroblasts internalize their class I molecules via uncoated cell surface vesicles and tubular invaginations when these molecules are cross-linked with multivalent ligands. T lymphocytes internalize their class I molecules spontaneously, but B lymphocytes do not internalize them at all. Here we describe a morphological investigation of the mechanism by which class I molecules are endocytosed by T lymphocytes. We show that, unlike fibroblasts, T lymphocytes spontaneously internalize 20-40% of their class I molecules in a process involving coated pits and coated vesicles. Thus, the endocytic pathway of class I molecules in T lymphocytes is similar to those of other more classical cell-surface receptors involved in receptor-mediated endocytosis. In contrast, the same class I molecules remained on the cell surface in B lymphocytes. These data show that class I molecules are differentially regulated in T and B lymphocytes and fibroblasts.
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http://dx.doi.org/10.1038/328724a0DOI Listing
September 1987

Phospholipid and arachidonic acid metabolism in zymosan-stimulated human monocytes: modulation by cAMP.

J Cell Physiol 1987 Jun;131(3):384-92

Receptor-ligand interaction in mononuclear phagocytes is intimately linked to alterations in membrane phospholipids and release of arachidonic acid (AA). In addition, synthesis of bioactive lipids from released AA can result in further modification of cell responses. Upon challenge with opsonized zymosan, [3H]-arachidonic acid ([3H]-AA)-labeled human monocytes released 25 +/- 2% of their incorporated radiolabel within 30 min. Pretreatment of the monocytes with 5 X 10(-4) M isobutylmethylxanthine (IBMX) or 1 X 10(-3) M dibutyryl cyclic AMP (d-cAMP) inhibited total [3H]-AA release in the presence of zymosan by 47% and 42%, respectively. Analysis of incorporated [3H]-AA in cellular phospholipid pools indicated that significant amounts of label were lost from both phosphatidylcholine (PC) and phosphatidylinositol (PI) during zymosan stimulation. Treatment with d-cAMP substantially inhibited the loss of label from PC, but had no affect on PI. HPLC analysis of cell supernatants from zymosan-treated cells indicated that 5-HETE was the predominant metabolite generated from [3H]-AA, and its production was depressed during treatment with d-cAMP. Phospholipase activity in human monocyte homogenates was not effected by d-cAMP or IBMX at the highest concentrations used, whether these were added directly to the homogenate or by pretreatment of whole cells, demonstrating that inhibition required an intact cell. These results suggest that human monocytes exposed to opsonized zymosan release AA via two mechanisms and that modulation by cAMP is indirectly effecting a phospholipase directed towards PC.
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http://dx.doi.org/10.1002/jcp.1041310310DOI Listing
June 1987

Characterization of an animal model of metastatic colon carcinoma.

Int J Cancer 1987 Feb;39(2):248-54

Although numerous animal tumor models have been used to study colon carcinoma, few display metastatic properties. We have characterized an animal tumor model that has 3 properties essential for the study of metastasis of colon carcinoma cells: epithelial cell origin; a reproducible pattern of metastatic behavior and the ability to be propagated both in vitro and in vivo to facilitate identification of biochemical correlates of metastasis. The K12/TR cell line was derived from a transplantable colon carcinoma induced by dimethylhydrazine in the BD-1X rat strain. Transmission electron microscopy of K12/TR cells demonstrated junctional complexes, desmosomes and surface microvilli characteristic of gastrointestinal epithelial cells. The epithelial cell origin of K12/TR was confirmed by demonstrating the presence of keratin, a marker of epithelial cells, but not vimentin, a constituent of mesenchymal cells. Secretion of CEA and Ca19-9 antigens by K12/TR cells in vitro was below the sensitivity of the assays (1 ng/ml and 6 U/ml respectively). K12/TR cells produced tumors following s.c. injection into syngeneic BD-1X rats, allogeneic RNU/rnuDF rats and xenogeneic CRL:nu/nuBR mice. Macroscopic lung metastases were observed in animals from all 3 groups. Distal lymph node metastases were more frequent in BD-1X rats than in nude rats or mice. The histological appearances of all tumors and metastases were similar, showing a moderate to poorly differentiated glandular carcinoma. Intrasplenic injections of K12/TR cells in nude mice resulted in liver colonization. Preferential growth of tumor cells at sites of trauma was also observed. The results show that the K12/TR system can be used as a model to study metastasis of colon carcinoma cells and may find utility in the testing of chemotherapeutic agents against metastatic lesions.
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http://dx.doi.org/10.1002/ijc.2910390221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7165865PMC
February 1987

Neutrophils may directly synthesize both H2O2 and O2- since surface stimuli induce their release in stimulus-specific ratios.

Inflammation 1985 Dec;9(4):425-37

Many stimuli induce neutrophils to undergo an oxidative burst and generate toxic oxygen metabolites. The major products are O2- and H2O2, the latter being presumed to arise by spontaneous dismutation of the former. If H2O2 were indeed derived exclusively from released O2- according to the equation 2O2- + 2H+----H2O2 + O2, one would expect that relationship to be reflected in the ratio of the two metabolites detectable in the extracellular mileu of stimulated neutrophils. A second corollary is that H2O2 should not form when cytochrome c is present to scavenge O2- before it can dismutate. Although H2O2 cannot be measured directly in the presence of cytochrome c because it is consumed in reoxidizing reduced cytochrome c, its presence can be detected indirectly by the ability of catalase to improve the apparent yield of reduced cytochrome c. We found that the relative amounts of extracellular H2O2 and O2- that could be measured in the environment of stimulated neutrophils varied with the stimulus and that catalase protected reduced cytochrome c from H2O2 oxidation when some stimuli were used but not with others. For example, the ratio of O2- to H2O2 produced by neutrophils exposed to PMA was about 2:1, the expected result if H2O2 were derived from O2-. However when cytochalasin B was added to the cells before the stimulus, the yield of H2O2 was reduced but not the yield of O2-. When cells were allowed to settle and spread on tissue culture plastic they produced equimolar amounts of O2- and H2O2. Coating the plastic with IgG doubled cytochrome c reduction without effecting H2O2. In contrast, coating with albumin reduced H2O2 without effecting cytochrome c reduction. Soluble IgG aggregates induced production of mostly O2- whereas immune complexes resulted in release of both metabolites. FMLP and A23187 were similar to the soluble IgG aggregates in their effects and induced release of proportionately more O2- than H2O2. The addition of catalase to the cytochrome c solution improved the yield of reduced cytochrome c when PMA or IgG was used to stimulate the cells but not when FMLP was used. These and other data suggest that H2O2 release is not a linear function of the amount of O2- generated and that either a variable fraction of O2- spontaneously dismutates to H2O2 or the neutrophil NADPH oxidase, in a manner analogous to xanthine oxidase, is capable, under some circumstances, of producing H2O2 as well as O2-.(ABSTRACT TRUNCATED AT 400 WORDS)
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http://dx.doi.org/10.1007/BF00916342DOI Listing
December 1985

Surface contact inhibits neutrophil superoxide generation induced by soluble stimuli.

Lab Invest 1985 May;52(5):515-22

Human neutrophils in suspension undergo a metabolic burst and generate reactive O2- metabolites upon exposure to many soluble and particulate stimuli. They can also be stimulated to produce O2- when in contact with surfaces. We found that when neutrophils were allowed to settle into protein-coated surfaces the amount of O2- they generated varied with the nature of the protein: IgG greater than bovine serum albumin greater than plastic greater than gelatin greater than serum greater than collagen. However, when polymorphonuclear leukocytes were permitted to settle onto a surface and then were stimulated with either phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine the O2- response was greatly diminished compared to control cells that were exposed to the stimulus in suspension. In contrast, superoxide production in response to the particulate stimulus opsonized zymosan was similar in both suspended and settled neutrophils. The degree of inhibition was not related to the degree of adherence since the diminished response occurred with all of the surfaces tested and in the presence of cytochalasin B. Onset of inhibition was very rapid as was recovery when cells were resuspended. Whereas production of O2- was greatly inhibited by surface contact, release of lysosomal enzymes was only slightly affected. The effect of surface contact did not appear to be mediated via activation of adenylate cyclase since the combination of a phosphodiesterase inhibitor and exogenous dibuteryl cyclic adenosine monophosphate did not inhibit phorbal myristate acetate O2- production, but surface contact did. These data indicate that surface contact such as would occur during diapedesis and chemotaxis profoundly alters neutrophil behavior by an unknown mechanism and imply that observations made on polymorphonuclear leukocytes in suspension cannot be generalized to polymorphonuclear leukocytes in tissue.
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May 1985

Colloidal lanthanum as a marker for impaired plasma membrane permeability in ischemic dog myocardium.

Am J Pathol 1975 May;79(2):207-18

Colloidal lanthanum salts have an average particle size of 40 degrees A; consequently, this electron-opaque marker remains extracellular and does not cross the intact plasma membrane. The affinity of lanthanum for calcium-binding sites on mitochondrial membranes makes it possible to demonstrate loss of plasma membrane integrity at the cellular level in ischemic myocardium. Biopsies were obtained from infarcted, marginal and normal areas 3 1/2 hours after ischemia was produced in 9 anesthetized closed-chest dogs by electrically induced thrombosis of the left anterior descending coronary artery. The tissue was immediately fixed in 4% glutaraldehyde and 0.1 M cacodylate buffer containing 1.3% La(NO3)3, pH 7.4, for 2 hours. In normal control tissue prepared this way the lanthanum tracer, as expected, was confirmed to the extracellular spaces, including, basement membranes, gap junctions and portions of the intercalated discs. Specimens taken near the center of frank infarctions all contained intracellular as well as extracellular lanthanum. Intracellular lanthanum could be seen evenly distributed around lipid droplets and in focal deposits around mitochondria. Only when mitochondria were disrupted did lanthanum gain access to internal sites on mitochondrial membranes. Areas marginal to the infarct contained cells in varying stages of degeneration including many that appeared normal by morphologic criteria alone. Intracellular lanthanum was present in many but not all of the marginal cells in which degenerative changes could be seen. Similarly a few of the cells that appeared morphologically normal contained intracellular lanthanum. The entry of lanthanum into some of these marginal cells and its exclusion from adjacent cells demonstrated that ischemic injury affects the permeability properties of the plasma membrane and independently of other intracellular morphologic changes and that lanthanum can be a sensitive indicator of such alteration in membrane permeability.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1912659PMC
May 1975

Cytochemical localization of lysosomal enzyme activity in normal and ischemic dog myocardium.

Am J Pathol 1975 May;79(2):193-206

The effect of ischemia on the integrity of myocardial lysosomes was observed 3 1/2 and 24 hours after the production of infarcts in 20 anesthetized closed-chest dogs by electrically induced thrombosis of the left anterior descending coronary artery. Biopsies from normal, marginal and infarcted areas were fixed and incubated to localize the lysosomal enzymes acid phosphatase and aryl sulphatase. Reaction product in normal cells was localized in small circular or oblong profiles between bundles of myofilaments and adjacent to mitochondria. In addition, curvilinear, membrane-bound profiles containing reaction product were found in close apposition to transverse tubules and near the free margins of the myocardial cells. Thus the distribution of elements of the sarcoplasmic reticulum. Additional reaction product was also seen in residual bodies, on myelin figures, and in the few conventional appearing spherical lysosomes. Little or no acid phosphatase or aryl sulphatase reaction product was seen in the sarcoplasmic reticulum of infarcted myocardium. The degree of cellular degeneration correlated with disappearance of enzyme activity from the sarcoplasmic reticulum and included disruption of membranes and loss of mitochondrial matrix and erosion of I but not A bands. Marginal areas showed variable amounts of cellular degeneration. Separation of myofilament bundles and loss of glycogen correlated with the localized disappearance of acid phosphatase and aryl sulphatase activity in marginal tissue. Disruption of mitochondrial and erosion of I bands correlated with extensive loss of these enzymes. The data suggest that degeneration of myocardial cells following ischemic injury is associated with release of endogenous lysosomal enzymes from the sarcoplasmic reticulum.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1912655PMC
May 1975
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