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Aim: To evaluate the gene expression changes that occur soon after the active infection of two susceptible cell types with human herpesvirus 8 (HHV-8). Materials & methods: The expression profile of 282 human genes involved in the inflammatory process was investigated in HHV-8 A1 or C3 subtype-infected and mock-infected human epithelial cells and lymphoid cells. Results: The HHV-8-induced transcriptional profiles in the epithelial and lymphoid cells were very different. A robust increase in the expression was found in genes belonging to different categories, especially the categories of inflammation response and signal transduction. Conclusion: These results indicate that during early infection, HHV-8 induces a variety of cell type-specific processes, thus providing infection signatures useful as potential targets for therapeutic intervention.

Viral infections have been associated with autoimmune connective tissue diseases. To evaluate whether active infection by Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus (HHV)-6, -7, -8, as well as parvovirus B19 (B19V) occur in patients with autoimmune connective tissue diseases, viral DNA loads were assessed in paired samples of serum and peripheral blood mononuclear cells (PBMCs) of 115 patients affected by different disorders, including systemic sclerosis, systemic, and discoid lupus erythematosus, rheumatoid arthritis, and dermatomyositis. Two additional groups, patients affected by inflammatory diseases (n=51) and healthy subjects (n=58) were studied as controls. The titers of anti-HHV-6 and anti-EBV antibodies were also evaluated. Cell-free HHV-6 serum viremia was detected in a significantly higher proportion of connective tissue diseases patients compared to controls (P

High-risk human papillomavirus (HR-HPV) genotype viral load and E6/E7 mRNA detection are proposed as surrogate markers of malignant cervical lesion progression. Currently, the use of commercially available DNA-based or mRNA-based tests is under investigation. In this study, the viral DNA load and E6/E7 mRNA detection of the five most common HR-HPV types detected in cervical cancer worldwide were compared in 308 cervical samples by using in-house type-specific quantitative real-time PCR assays and PreTect HPV-Proofer test, respectively. Sensitivity and negative predictive values were higher for the HPV-DNA assays combined (95.0% and 96.0%, respectively) than the RNA assays (77.0% and 88.0%, respectively); conversely, the mRNA test showed a higher specificity and higher positive predictive value (81.7% and 66.9%, respectively) than the DNA test (58.6% and 52.5%, respectively) for detecting histology-confirmed high-grade cervical intraepithelial neoplasia. A significantly higher association between viral DNA load and severity of disease was observed for HPV 16 and 31 (γ = 0.62 and γ = 0.40, respectively) than for the other HPV types screened. A good degree of association between the two assays was found for detection of HPV 16 (k = 0.83), HPV 18 (k = 0.72), HPV 33 (k = 0.66), and HPV 45 (k = 0.60) but not for HPV 31 (k = 0.24). Sequence analysis in L1 and E6-LCR regions of HPV 31 genotypes showed a high level of intra-type variation. HR-HPV viral DNA load was significantly higher in E6/E7 mRNA positive than negative samples (P < 0.001), except for HPV 31. These findings suggest that transcriptional and replicative activities can coexist within the same sample

The demogenetic structure of the Amiata donkey, an endangered breed from Central Italy, was investigated using information from pedigrees. Genealogical data of 602 donkeys reared in Tuscany were recorded in a database and analysed by the computer package ENDOG. Population size increased from 89 subjects in 1995 to 503 (129 males and 374 females) in 2005. Animals were distributed among 152 herds, but the effective number of herds was 21, suggesting that a small number of herds provided stallions for the entire breed. The maximum number of traced generation was 4, the mean maximum generation was 1.14, the mean com- plete generation was 0.53, and the mean equivalent generation was 0.78. The average relatedness coeffi- cient (AR) in the 503 alive animals was 0.94% while the mean F was 0.29% so the effective population size was 172.41. Among 24 animals with a 4-generation history, 3 (12.5%) were 25% inbred. Although the incompleteness of genealogical information did not permit accurate inference of the current values of popu- lation genetic parameters, the present work represents a first step towards an efficient management of the breed.

Liposomes containing Distamycin A (DA) may be clinically useful in the treatment of ocular HSV infections, especially in acyclovir-resistant HSV keratitis. This study evaluated the in vitro and in vivo performance of a topical controlled release liposomal formulation containing DA (DA-Lipo) aimed at reducing the toxicity of the encapsulated active agent and improving drug uptake by ocular tissues. The bioavailability of DA in the tear fluid and the DA uptake into the cornea were increased after instillation of DA-Lipo in rabbits, reaching the DA corneal concentration corresponding to IC50 values against HSV without any sign of transcorneal permeation of drug. DA-Lipo was definitely less cytotoxic then plain DA in rabbit corneal epithelial cells. These results provide new insights into the correlation between the in vitro data and the drug kinetics following ocular applications of liposomal vesicles.

The purpose of this study was the output and set up of the milk array, a dedicated array designed to investigate the expression levels of many genes involved in cow mammary gland inflammation and milk production regulation. First, a new targeted genes panel was selected. Successively, the microarray reliability was examined by yellow and dye swap experiments using the normal and mastitic mammary gland samples from the same cow. The sensitivity and reliability were evaluated using different amounts of the same mastitic mammary gland RNA: a good linear regression (R 2 = 0.758) was obtained also using only 3 μg of RNA. We used both reverse transcriptase RT-qPCR and the microarray to analyze 100 bovine genes (96 known to be involved in inflammation and milk production regulation and four housekeeping genes) in pooled total RNA isolated from tissue samples. All genes were detectable by RT-qPCR and microarray: a good mean correlation coefficient over all samples of 0.885 showed that both methods were similarly well suited to analyze gene expression in these samples. This report describes the development of small DNA microarray of fully defined genes suitable for analysis of expression of many genes involved in cow mammary gland inflammation and milk production regulation; this platform will prove useful as diagnostic tool prototype to perform a more in-depth analysis of the milk quality and mammary glands health status. © 2012 Springer Science+Business Media New York.

© 2012 Matteoli and Ceccherini-Nelli, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Viral DNA and cDNA Array in the Diagnosis of Respiratory Tract Infections

Genital herpes is caused by herpes simplex virus 1 (HSV-1) and HSV-2, and its incidence is constantly increasing in the human population. Regardless of the clinical manifestation, HSV-1 and HSV-2 infections are highly transmissible to sexual partners and enhance susceptibility to other sexually transmitted infections. An effective vaccine is not yet available. Here, HSV-1 glycoprotein B (gB1) was delivered by a feline immunodeficiency virus (FIV) vector and tested against HSV-1 and HSV-2 vaginal challenges in C57BL/6 mice. The gB1 vaccine elicited cross-neutralizing antibodies and cell-mediated responses that protected 100 and 75% animals from HSV-1- and HSV-2-associated severe disease, respectively. Two of the eight fully protected vaccinees underwent subclinical HSV-2 infection, as demonstrated by deep immunosuppression and other analyses. Finally, vaccination prevented death in 83% of the animals challenged with a HSV-2 dose that killed 78 and 100% naive and mock-vaccinated controls, respectively. Since this FIV vector can accommodate two or more HSV immunogens, this vaccine has ample potential for improvement and may become a candidate for the development of a truly effective vaccine against genital herpes.

The study was performed to determine if there is an association between the genotype and transmission of HHV-8 types A and C. These HHV-8 subtypes are prevalent in the area of North of Sardinia, which is an island off west Italy's mainland that has a high HHV-8 seroprevalence (35%). Blood and saliva samples from 30 patients with classic Kaposi's sarcoma who were lifetime residents of North Sardinia were analyzed to identify the HHV-8 genotype and quantitate the viral load. Genotype A, especially A1 subtype, was found more frequently (9/30 patients) and had a significantly higher viral load in saliva compared to blood (P = 0.029), where type C was found more frequently but with a viral load lower than 103 copies/ml. To determine if there is a correlation between the viral genotype and cellular tropism, type A1 and C3 HHV-8 viral particles were obtained from cell lines BCBL1 and BC3 infected chronically with HHV-8 A1 and C3 genotypes respectively and used to infect HEK293 epithelial origin cells and PBMCs in vitro. The data indicate that the A1 HHV-8 genotype is tropic and replicates at higher levels in the epithelial cell lines. J. Med. Virol. 84:786–791, 2012. © 2012 Wiley Periodicals, Inc.

The dramatic size contraction of local cattle breeds due to replacement with cosmopolite improved breeds highlights the need for native genetic resources conservation. In 1985, the Anagraphic Register of local cattle breeds and small-size ethnic groups was established by the Italian Ministry of Agriculture and Forestry. Calvana, Pontremolese and Garfagnina are among the included breeds. They are all native from Tuscany. Present breeding area covers the provinces of Firenze, Prato, Pistoia and Siena for the Calvana breed (around 280 heads), while it is restricted to the province of Lucca for both Garfagnina (around 180 heads) and Pontremolese. This latter breed consists, nowadays, of less than 40 heads, while being around 15000 in 1940s.The characterization of the genetic structure and variability via molecular markers could provide useful information for breed management and conservation. In the present study, a total of 149 animals, evenly distributed among the three breeds, were genetically char...

Background: Little is known about the involvement of human herpesviruses 6 and 7 (HHV-6 and HHV-7) in autoimmune connective tissue diseases (ACTD). Objective: To determine the prevalence of active infection with HHV-6 and HHV-7 in patients with ACTD. Study design: The presence and quantity of HHV-6 DNA was determined by quantitative real-time PCR in a cross-sectional study of serum, peripheral blood mononuclear cells, and tissues obtained from 58 ACTD patients and 38 healthy subjects (HS). Specific anti-HHV-6 antibody titer was also measured. Results: HHV-6 serum viremia occurred in a significantly higher proportion of ACTD patients compared to HS [26/58 (44.8%) vs. 1/38 (2.6%), p = 0.001] with the highest reactivation frequency [7/10 (70%)] observed in patients with scleroderma. Moreover, HHV-6 in serum was associated with ACTD activity (22/38 vs. 4/20, p < 0.05). Higher titers of HHV-6 antibodies were found in ACTD patients than in HS, although HHV-6 seroprevalence among patients with ACTD and HS was similar. HHV-7 viremia was not detected in any patients or HS controls. Conclusion: The frequent reactivation of HHV-6 in scleroderma and other ACTD, especially when active, suggests that HHV-6 may play a role in the pathogenesis of these diseases. © 2009 Elsevier B.V. All rights reserved

The results of the pulsed-field gel electrophoresis and the sequence-based typing (using the loci flaA, pilE, asd, mip, mompS and proA) were compared for subtyping of Legionella pneumophila 1 strains isolated from a hospital water supply. Molecular typing was carried out on 61 isolates (38% of the positive samples) selected on space and temporal criteria in order to follow the evolution of the water-system colonization. For all the 61 isolates, the sequence of the amplified mip gene fragment identified Legionella pneumophila strain Wadsworth. Genotype testing by PFGE analysis showed three different patterns, correspondent to three SBT types according to the allelic profiles. Both PFGE and SBT indicated the circulation and the persistence in the hospital potable water-system of three types randomly distributed in space and time. The two molecular methods adopted showed a 100% concordance, although a low degree of genetic heterogeneity characterized the isolates. The electrophoretic patterns were sufficiently unambiguous to consider PFGE a highly discriminatory typing method, but the SBT technique besides accurately characterizing isolates, was able to identify Legionella strains through analysis of the mip gene. A typing method with this level of discriminatory power has great potential for assisting in epidemiological studies.

Objective(s): Herpes simplex virus (HSV) infections in immunocompromised individuals may require prolonged antiviral therapy resulting in the emergence of viral strains resistant to the currently employed antiviral drugs. Distamycin A (DA), a basic antibiotic belonging to the lexitropsin DNA minor groove binding drugs, exhibits antiviral properties. In this study we evaluated the in vitro cytotoxicity and antiviral activity of DA against HSV type 1 and HSV type 2 clinical isolates from transplanted patients and compared them with those of acyclovir (ACV) in search of alternative antiviral drugs. Methods: Viral detection and typing was performed by multiplex PCR and immunofluorescence assay; the in vitro cytotoxicity of DA and the antiviral activity of ACV and DA was evaluated respectively by neutral red uptake assay and plaque reduction assay for HSV2 isolates and fluorescence reduction assay for HSV1 isolates. Results: Tissue culture 50% cytotoxic concentration of DA was 58 µM. Tissue culture 50% inhibitory concentration values ranged from 0.16 to 7.4 µM for the ACV-sensitive and from 5.4 to 32 µM for the ACV-resistant viral strains. Conclusions: In spite of the lower activity against ACV-resistant strains, DA may be used as an antiherpetic drug.

The abdomino-cruroplasty is the result of a combination of an abdominoplasty (or "mini-lift" as described by French authors) and a reduction plasty of the thighs. Our prime objective is the one-stage correction of the cutaneous and fatty excess of both the abdomen and the thighs with a single scar hidden in the skin folds; we also obtain an equilibration of "up" and "down" forces to avoid deformation of the pubic region. The technical points are explained.