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Little is known about the impact of space (geography/ancestry) and time (age of the individuals) on DNA methylation variability in humans. We investigated DNA methylation of the imprinted IGF2/H19 locus in: i) a cohort of individuals homogeneous for age and gender (males with restricted age range: 30-50 years) belonging to four Italian districts representative of the major genetic clines, informative for the geographical dimension; ii) a cohort of monozygotic (MZ) and dizygotic (DZ) twins of different ages (age-range: 22-97 years), informative for the temporal dimension. DNA methylation of the analyzed regions displayed high levels of inter-individual variability that could not be ascribed to any geographical cline. In MZ twins we identified two IGF2/H19 regions where the intra-couple variations significantly increased after the age of 60 years. The analysis of twins' individual life histories suggests that the within twin pairs difference is likely the result of the aging process itself, as sharing a common environment for long periods had no effect on DNA methylation divergence. On the whole, the data here reported suggest that: i) aging more than population genetics is responsible for the inter-individual variability in DNA methylation patterns in humans; ii) DNA methylation variability appears to be highly region-specific.

Objective: To investigate the costimulatory role of Crry/p65 (Crry), a membrane complement regulatory protein, on the expansion and function of natural Treg cells and their ability to ameliorate proteoglycan-induced arthritis (PGIA), an animal model of inflammatory arthritis in which the role of natural Treg cells is not well established., Methods: CD4+CD25+ natural Treg cells from BALB/c mice were activated in vitro and costimulated by Crry. The expanded cells were phenotypically characterized, and their suppressive effect on T cell proliferation was assayed in vitro. The potential prophylactic and therapeutic effects of this population versus those of natural Treg cells in PGIA were studied. The clinical score, histology, the antigen-specific isotype antibody pattern, in vitro T cell responses, and the presence of Treg cells in the paws were studied., Results: Crry costimulation enhanced the in vitro expansion of natural Treg cells while maintaining their phenotypic and suppressive properties. Crry-expanded Treg cells had stronger suppressive properties in vivo and a longer ameliorating effect in the PGIA model than did natural Treg cells. Crry-expanded Treg cells suppressed T cell- and B cell-dependent responses in PGIA, changing the pathogenic antibody isotype pattern and decreasing antigen-dependent secretion of cytokines, including interferon-γ, interleukin-12 (IL-12), and IL-17. Increased FoxP3 expression was detected in the paws of mice transferred with Crry-expanded Treg cells., Conclusion: Crry-mediated costimulation facilitates in vitro expansion of natural Treg cells while maintaining their suppressive properties in vitro and in vivo in the PGIA model. These results highlight the potential of the complement regulatory protein Crry to costimulate and expand natural Treg cells capable of suppressing disease in an animal model of chronic inflammatory arthritis., (Copyright © 2011 by the American College of Rheumatology.)

Rifaximin, a semisynthetic, rifamycin-based non-systemic antibiotic is used in the treatment of acute and chronic gastrointestinal disorders. The aim of this study was the elucidation of the molecular structure of the 802 Dalton impurity which was found in Rifaximin industrial batches and reported with an erroneous structure in European Pharmacopoeia 6.5 (2009) [7] monograph as Rifaximin Impurity H. This impurity was isolated from Rifaximin by preparative HPLC and purified by column chromatography. The molecular structure was evidenced by means of (1)H and (13)C NMR spectroscopy, mass spectrometry and FT-IR., (Copyright 2009 Elsevier B.V. All rights reserved.)

A study was undertaken to determine the cardioprotective effects of parrodienes prepared from the feather pigments of parrots (Ara macao). Adult male Sprague-Dawley rats were divided into three experimental groups and perfused with KHB buffer with or without treatment of 2,4,6-octatrienal (1) (50 mM) or its potassium salt (2) (50 mM). All hearts were then subjected to 30 min ischemia followed by a 2 h reperfusion. Ischemia/reperfusion resulted in a significant amount of tissue injury, cardiomyocyte apoptosis, and depression in hemodynamic functions. Parrodiene treatment prevented the development of myocardial injury after ischemia/reperfusion. Western blot analysis indicated that 1 and 2 reduced the oxidative stress, induced the expression of Bcl-2, and caused increased phosphorylation of Akt. These agents also reduced myocardial ischemic reperfusion injury presumably by reducing oxidative stress and activating the survival signal through the Akt-Bcl-2 pathway.

Budesonide is the 16alpha,17alpha-acetal of 16alpha-hydroxyprednisolone with n-butyraldehyde, endowed with anti-inflammatory activity. In a sample of budesonide tablets, kept for 3 years at 25 degrees C and 60% RH unknown impurities, not reported in European Pharmacopoiea, were present. Their identification was achieved by means of chemical and spectroscopic methods.

The inducible co-stimulator (ICOS, CD278) is essential to the efficient development of normal and pathological immune reactions. Since ICOS-deficient mice have enhanced susceptibility to experimental allergic encephalomyelitis (EAE), we have functionally analyzed a CD4+ICOS+ population comprising 6-15% of all CD4+ T cells in secondary lymphoid organs of unmanipulated wild-type mice and checked for their ability to suppress EAE. In C57BL/6 mice, CD4+ICOS+ cells were a major source of cytokines including IFN-gamma, IL-2, IL-4, IL-10 or IL-17A. Upon activation, these cells showed preferentially enhanced production of IL-4 or IL-10 but inhibited IFN-gamma production. In contrast, CD4+ICOS- cells mainly produced IFN-gamma. Interestingly, CD4+ICOS+ cells partially suppressed the proliferation of CD4+ICOS- or CD4+CD25- lymphocytes 'in vitro' by an IL-10-dependent mechanism. Furthermore, CD4+ICOS+ activated and expanded under appropriate conditions yielded a population enriched in cells producing IL-10 and T(h)2 cytokines that also suppressed the proliferation of CD4+CD25- lymphocytes. CD4+ICOS+ cells, before or after expansion in vitro, reduced the severity of EAE when transferred to ICOS-deficient mice. In the same EAE model, lymph node cells from ICOS-deficient mice receiving ICOS+ cells showed reduced IL-17A production and enhanced IL-10 secretion upon antigen activation in vitro. Thus, naturally occurring CD4+ICOS+ cells, expanded or not in vitro, are functionally relevant cells able of protecting ICOS-deficient mice from severe EAE.

Unknown by-product in Simvastatin synthesis from Lovastatin was found. The elucidation of this molecular structure by means of (1)H and (13)C NMR spectroscopy, HPLC/MS, MS/MS and FT-IR was shown. The mentioned by-product, originated during Merck Sharp and Dhome synthesis scheme was isolated in the second-last step replacing butylamine with benzylamine. The spectroscopic results agreed with a molecular formula C(32)H(43)NO(3). The proposed structure of this compound, characterised by the presence of a conjugated dienic system in the heptanoic acid amide residue, was alpha,beta,gamma,delta unsaturated Simvastatin N-benzylamide.

The purpose of this study is the development of a quantification method to detect the amount of amorphous cyclosporine using Fourier transform infrared (FTIR) spectroscopy. The mixing of different percentages of crystalline cyclosporine with amorphous cyclosporine was used to obtain a set of standards, composed of cyclosporine samples characterized by different percentages of amorphous cyclosporine. Using a wavelength range of 450-4,000 cm(-1), FTIR spectra were obtained from samples in potassium bromide pellets and then a partial least squares (PLS) model was exploited to correlate the features of the FTIR spectra with the percentage of amorphous cyclosporine in the samples. This model gave a standard error of estimate (SEE) of 0.3562, with an r value of 0.9971 and a standard error of prediction (SEP) of 0.4168, which derives from the cross validation function used to check the precision of the model. Statistical values reveal the applicability of the method to the quantitative determination of amorphous cyclosporine in crystalline cyclosporine samples.

Crry/p65 is a type I glycoprotein, which protects mouse T cells from complement attack. We have previously shown that complement receptor I-related protein Crry/p65 (Crry) ligation has a costimulatory effect on mouse CD4+ T cell activation. Here, we have examined the mechanisms responsible for Crry costimulation, addressing the question of whether Crry potentiates signal transduction starting at the T cell receptor (TCR)/CD3 complex or promotes distinct costimulatory signals. We show that Crry increases early TCR-dependent activation signals, including p56lck-, zeta-associated protein-70 (ZAP-70), Vav-1, Akt, and extracellular signal-regulated kinase (ERK) phosphorylation but also costimulation-dependent mitogen-activated protein kinases (MAPK), such as the stress-activated c-Jun N-terminal kinase (JNK). It is intriguing that Crry costimulus enhanced p38 MAPK activation in T helper cell type 1 (Th1) but not in Th2 cells. A fraction of Crry is found consistently in the detergent-insoluble membrane fraction of Th1 or Th2 cells or CD4+ lymphoblasts. Crry costimulation induced clustering of lipid rafts, increasing their content in Crry, CD3epsilon, and p59-60 forms of p56lck, and caused actin polymerization close to the site of activation in Th2 cells. Such events were inhibited by wortmannin, suggesting a role for phosphatidylinositol-3 kinase in these effects. The Crry cytoplasmic domain was required for JNK activation and interleukin-4 secretion but not for the presence of Crry in rafts or activation of p56lck, ZAP-70, Akt, Vav-1, or ERK. This suggests that Crry costimulation involves two different but not mutually exclusive signal transduction modules. The dual function of Crry as a complement regulatory protein and as a T cell costimulator illustrates the importance of complement regulatory proteins as links between innate and adaptive immunity.

A new chemical named parrodienes has been extracted from parrots' plumage. From the chemical point of view, parrodienes are polyunsaturated aldehydes similar to carotenoids. On the basis of this similarity we organized some biological experiments to evaluate the inhibition of lipoperoxidation of cell membranes induced by CCl4, protection against ultraviolet rays, anti-inflammatory activity and protection from an increase of ornithine-decarboxylase as marker of tumoral skin alteration. The results of these experiments showed that unsaturated dienes (parrodienes) play an important role on the inhibition and prevention of many biological processes that are at the basis of different pathological disorders.

Moving from the interest as immunomodulatory agent of ST789 was studied the synthesis of series of N9alkylated hypoxanthine and adenine. The synthesis and the chemical physical properties of these derivatives are here described.

Carotenoids exert immunomodulating, immunostimulating, and antioxidant actions in mammals and are major determinants of coloration in animals. Honest advertisement models of sexual selection propose that male ornaments, including coloration, are reliable indicators of male quality. Because of their simultaneous effects on male coloration and immunity, carotenoids might mediate the hypothesized relationship between the expression of epigamic coloration and parasitism in vertebrates. We analyzed the relationship between immune profile and concentration of lutein, the most abundant carotenoid in the plasma of male barn swallows (Hirundo rustica). Consistent with our predictions, lutein plasma concentration was negatively correlated with gamma-globulin plasma levels and concentration of selected leukocyte types in peripheral blood, suggesting that, to exert immune function, carotenoids are taken up from plasma, thus becoming unavailable for epigamic signaling. The coloration of red feathers of the throat of adult males was positively related to plasma concentration of lutein, but not with immunologic variables, consistent with the idea that more brightly colored males do not pay a larger immunological cost for their coloration compared with less brightly colored males. Length of male tail ornaments, which is currently under directional sexual selection, was positively correlated with lutein plasma levels. In species where carotenoids limit immune function, demands for pigments for sexual signaling might compete with those for immunity, thus generating a mechanism that enforces honesty on the signal.

GC/ms is an important method to determine drugs in biological fluids. For each of the most commonly used drugs a methanolic solution (100 micrograms/ml) was prepared; 30 microliters of each solution was evaporated using nitrogen flow and than diluted in 100 microliters of a derivatizing mixture constituted by MSTFA and toluene (30/70). After a short reaction time at room temperature 1 ml of each solution was injected into a SE-52 column (15 m x 0.25 mm) using chemical ionization with acetonitrile. This method has a good signal/noise ratio and is very useful in screening techniques.

The natural history of cervical intraepithelial neoplasia (CIN) evidenced in such pathologies a different biological behaviour due to the presence of similar morphological atypia with different potential development. To biologically characterize CIN lesions we valued by flow cytometry, the DNA content and the proliferative activity of 53 biopsies obtained by colposcopy. Aneuploid histograms were present in none of the histologically negative lesions, in 14% of the CIN I, in none CIN II, and in 40% of the CIN III. The mean value of S-phase was 2.7% in non dysplastic lesions, 5% in the CIN I, 2.5% in the CIN II and 6.1% in the CIN III. In this study the findings of aneuploid cells and the value of proliferative activities seems to be not correlated with the histological features. The clinical and instrumental follow up of the considered patients could establish the eventual prognostic significance of cytofluorimetric parameters in CIN lesions.

The synthesis and chemical and physical properties of some N2-[omega-(pyrimidin-1-yl)carboxyalkyl]-L-arginines are here described. Experiments of immunopharmacological evaluations are also described.

Aims and Background: The characterization of pleural metastases from lung adenocarcinoma is often limited to single biologic features., Methods: The present paper describes the cellular kinetic parameters, as well as immunocytochemical, ultrastructural and genetic characteristics of the new DV90 cell line, established from the pleural effusion of a stage IV lung adenocarcinoma., Results: The cell line has a diploid DNA content, a doubling time of 24 h and 7% cloning efficiency, it is tumorigenic in nude mice. Ultrastructural investigation revealed the typical features of lung adenocarcinoma; the diagnosis was confirmed by its immunohistochemical reactivity with a panel of monoclonal antibodies specifically capable of identifying adenocarcinoma cells. Genetic analysis revealed a 46 X, -Y, +8, der (6)t(6?)(q27;?) karyotype and hyperexpression of the protein codified by genes Her2/Neu and p53., Conclusion: The importance of multidisciplinary biologic characterization in identifying the origin and biological behavior of pleural metastases deriving from lung adenocarcinoma is discussed.

We have considered the colpo-cytologic characteristic of 83 patients with CIN II-III histologic lesions over 900 colposcopic biopsies carried out at our Department from 1990-1992. In particular 38 cases were classified as CIN II of which 23 associated with HPV cytopathic feature, while 45 cases were, classified as CIN III, of which 13 associated with HPV c.f. 29% of CIN II were evident in women under 30 years of age; in this group the age decreased with the presence of HPV. 31% of CIN III were present in women under 35. A good correlation between cytologic and histological analysis on the same patient was observed particularly in CIN with the higher grade. Also a good correlation between colposcopy grading and CIN was observed. In CIN II, grade I images were present, while in CIN III, punctuation and white epithelium were the most common features. Our study shows also the impossibility of distinguishing between the images of simple viral infection and their related CIN morphologic patterns. Colposcopy represents a basic test for the definition of CIN, particularly for those with higher grade, and a complementary test for the definition of the topography of the lesions with the correct choice of the therapeutic treatment.

We describe the production of human granulocyte-macrophage colony-stimulating factor (hGM-CSF), by cell lines established from patients with different stages of breast, lung and colon adenocarcinoma. GM-CSF expression has been identified by immunocytochemistry determination, quantified on conditioned medium with specific ELISA procedure and evaluated by means of proliferation and differentiation of normal human monocytic and granulocytic progenitors. The growth of cell lines after incubation with exogenous GM-CSF and antibody-antiGM-CSF was not modified. To better understand the patho-physiologic role of hGM-CSF in vivo we also estimated its serum levels at diagnosis in 75 patients with breast lung and colon adenocarcinoma and in 69 healthy person. Only two patients showed detectable GM-CSF levels. The lack of growth modulation observed in vitro with exogenous GM-CSF and antibody anti-GM-CSF suggests a non autocrine secretion by adenocarcinoma cells. The serum investigation evidences that the leukocytosis observed in adenocarcinoma patients is unrelated to a GM-CSF constitutive tumor production in vivo.

The state of the fundus oculi was investigated in 40 insulin-dependent and 20 non-insulin dependent diabetics. In both groups the state of the fundus oculi was correlated with total cholesterol, LDL, HDL, glycosylated haemoglobin, platelet adhesiveness, triglycerides and duration of the disease. In the case of insulin-dependent diabetics a correlation was also sought with daily doses of insulin. The fundus oculi was normal in 21/40 insulin-dependent and 14/20 non-insulin-dependent subjects. Multiple regression showed a positive correlation between insulin-HbA doses and duration of the disease and the fundus oculi. Simple liner regression showed a negative correlation between insulin doses and fundus, a tendentially positive correlation between platelet adhesion and fundus. A positive correlation between LDL-platelet adhesion and fundus was also shown in the non-insulin-dependent subjects. These results suggest that factors held to influence macroangiopathy also have an effect on macroangiopathy, that the check up of diabetics is important for the prevention of retinopathy, that insulin doses should be strictly limited to the necessary amount for the control of diabetes since excess is as harmful as insufficiency.

Biosynthetic Human Insulin (BHI) was administered to 20 diabetics for six months. A clinical examination, glycaemia before and after eating, total glycosuria-ketonuria, and glycosylated haemoglobin tests were performed at fixed intervals, and the results were statistically analysed. It is concluded that human insulin produces no significant side effects. More important that the type of insulin treatment is effective outpatients surveillance of the diabetic, to ensure an adequate metabolic compensation.