Inhomogeneous Diastereomeric Composition of Mongersen Antisense Phosphorothioate Oligonucleotide Preparations and Related Pharmacological Activity Impairment

Mongersen is a 21-mer antisense oligonucleotide designed to downregulate Mothers against decapentaplegic homolog 7 (SMAD7) expression to treat Crohn's disease. Mongersen was manufactured in numerous batches at different scales during several years of clinical development, which all appeared identical, using common physicochemical analytical techniques, while only phosphorous-31 nuclear magnetic resonance (P-31-NMR) in solution showed marked differences. Close-up analysis of 27 mongersen batches revealed marked differences in SMAD7 downregulation in a cell-based assay. Principal component analysis of P-31-NMR profiles showed strong correlation with SMAD7 downregulation and, therefore, with pharmacological efficacy in vitro. Mongersen contains 20 phosphorothioate (PS) linkages, whose chirality (Rp/Sp) was not controlled during manufacturing. A different diastereomeric composition throughout batches would lead to superimposable analytical data, but to distinct P-31-NMR profiles, as indeed we found. We tentatively suggest that this may be the origin of different biological activity. As similar manifolds are expected for other PS-based oligonucleotides, the protocol described here provides a general method to identify PS chirality issues and a chemometric tool to score each preparation for this elusive feature

Circulating endothelial progenitor cells are increased in patients with classic Kaposi’s Sarcoma

Accumulating evidence indicates that tumor angiogenesis is supported by the mobilization and incorporation of endothelial progenitor cells (EPCs), highly proliferative elements derived from the bone marrow. EPCs have been detected at increased frequency in the circulation of patients with different types of cancer. Circulating EPCs have never been quantified in patients with Kaposi’s sarcoma (KS), an angioproliferative malignancy characterized by typical spindle-shaped tumor cells of endothelial origin. In this study, we analyzed the number of EPCs in the peripheral blood of 29 patients with classic KS (cKS) compared with 27 matched healthy controls. EPCs were measured by flow cytometry on fresh blood samples at a single time point. The number of circulating EPCs was significantly higher in cKS patients than controls. EPC count did not correlate with the plasmatic levels of the main proangiogenic factors possibly involved in EPC proliferation and mobilization, thus suggesting that the increased number of EPCs in cKS patients may rather be related to direct or indirect effects of viral environment sustained by HHV-8, the causative agent for KS. The increase of EPCs was significantly more pronounced in cKS patients with slowly evolving than rapidly evolving disease, likely reflecting a localization of EPCs within the lesions during the active phases of KS. Overall, this study demonstrates that EPCs are increased in the peripheral blood of cKS patients and may suggest that changes in the number of circulating EPCs may predict disease progression and may therefore be proposed as a biomarker in the follow-up of KS patients

Human Herpesvirus-8 Infection Leads to Expansion of the Preimmune/Natural Effector B Cell Compartment

BACKGROUND: Human herpesvirus-8 (HHV-8) is the etiological agent of Kaposi's sarcoma (KS) and of some lymphoproliferative disorders of B cells. Most malignancies develop after long-lasting viral dormancy, and a preventing role for both humoral and cellular immune control is suggested by the high frequency of these pathologies in immunosuppressed patients. B cells, macrophages and dendritic cells of peripheral lymphoid organs and blood represent the major reservoir of HHV-8. Due to the dual role of B cells in HHV-8 infection, both as virus reservoir and as agents of humoral immune control, we analyzed the subset distribution and the functional state of peripheral blood B cells in HHV-8-infected individuals with and without cKS. METHODOLOGY/PRINCIPAL FINDINGS: Circulating B cells and their subsets were analyzed by 6-color flow cytometry in the following groups: 1- patients HHV-8 positive with classic KS (cKS) (n = 47); 2- subjects HHV-8 positive and cKS negative (HSP) (n = 10); 3- healthy controls, HHV-8 negative and cKS negative (HC) (n = 43). The number of B cells belonging to the preimmune/natural effector compartment, including transitional, pre-naïve, naïve and MZ-like subsets, was significantly higher among HHV-8 positive subjects, with or without cKS, while was comparable to healthy controls in the antigen-experienced T-cell dependent compartment. The increased number of preimmune/natural effector B cells was associated with increased resistance to spontaneous apoptosis, while it did not correlate with HHV-8 viral load. CONCLUSIONS/SIGNIFICANCE: Our results indicate that long-lasting HHV-8 infection promotes an imbalance in peripheral B cell subsets, perturbing the equilibrium between earlier and later steps of maturation and activation processes. This observation may broaden our understanding of the complex interplay between viral and immune factors leading HHV-8-infected individuals to develop HHV-8-associated malignancies

Randomized trial on the effects of a combined physical/cognitive training in aged MCI subjects: the Train the Brain study

Age-related cognitive impairment and dementia are an increasing societal burden. Epidemiological studies indicate that lifestyle factors, e.g. physical, cognitive and social activities, correlate with reduced dementia risk; moreover, positive effects on cognition of physical/cognitive training have been found in cognitively unimpaired elders. Less is known about effectiveness and action mechanisms of physical/cognitive training in elders already suffering from Mild Cognitive Impairment (MCI), a population at high risk for dementia. We assessed in 113 MCI subjects aged 65-89 years, the efficacy of combined physical-cognitive training on cognitive decline, Gray Matter (GM) volume loss and Cerebral Blood Flow (CBF) in hippocampus and parahippocampal areas, and on brain-blood-oxygenation-level-dependent (BOLD) activity elicited by a cognitive task, measured by ADAS-Cog scale, Magnetic Resonance Imaging (MRI), Arterial Spin Labeling (ASL) and fMRI, respectively, before and after 7 months of training vs. usual life. Cognitive status significantly decreased in MCI-no training and significantly increased in MCI-training subjects; training increased parahippocampal CBF, but no effect on GM volume loss was evident; BOLD activity increase, indicative of neural efficiency decline, was found only in MCI-no training subjects. These results show that a non pharmacological, multicomponent intervention improves cognitive status and indicators of brain health in MCI subjects

Brain hemodynamic intermediate phenotype links Vitamin B12 to cognitive profile of healthy and mild cognitive impaired subjects

Vitamin B12, folate, and homocysteine are implicated in pivotal neurodegenerative mechanisms and partake in elders' mental decline. Findings on the association between vitamin-related biochemistry and cognitive abilities suggest that the structural and functional properties of the brain may represent an intermediate biomarker linking vitamin concentrations to cognition. Despite this, no previous study directly investigated whether vitamin B12, folate, and homocysteine levels are sufficient to explain individual neuropsychological profiles or, alternatively, whether the activity of brain regions modulated by these compounds better predicts cognition in elders. Here, we measured the relationship between vitamin blood concentrations, scores at seventeen neuropsychological tests, and brain activity of sixty-five elders spanning from normal to Mild Cognitive Impairment. We then evaluated whether task-related brain responses represent an intermediate phenotype, providing a better prediction of subjects' neuropsychological scores, as compared to the one obtained considering blood biochemistry only. We found that the hemodynamic activity of the right dorsal anterior cingulate cortex was positively associated (p value < 0 05 cluster corrected) with vitamin B12 concentrations, suggesting that elders with higher B12 levels had a more pronounced recruitment of this salience network region. Crucially, the activity of this area significantly predicted subjects' visual search and attention abilities (p value = 0 0023), whereas B12 levels per se failed to do so. Our results demonstrate that the relationship between blood biochemistry and elders' cognitive abilities is revealed when brain activity is included into the equation, thus highlighting the role of brain imaging as intermediate phenotype.Vitamin B12, folate, and homocysteine are implicated in pivotal neurodegenerative mechanisms and partake in elders' mental decline. Findings on the association between vitamin-related biochemistry and cognitive abilities suggest that the structural and functional properties of the brain may represent an intermediate biomarker linking vitamin concentrations to cognition. Despite this, no previous study directly investigated whether vitamin B12, folate, and homocysteine levels are sufficient to explain individual neuropsychological profiles or, alternatively, whether the activity of brain regions modulated by these compounds better predicts cognition in elders. Here, we measured the relationship between vitamin blood concentrations, scores at seventeen neuropsychological tests, and brain activity of sixty-five elders spanning from normal to Mild Cognitive Impairment. We then evaluated whether task-related brain responses represent an intermediate phenotype, providing a better prediction of subjects' neuropsychological scores, as compared to the one obtained considering blood biochemistry only. We found that the hemodynamic activity of the right dorsal anterior cingulate cortex was positively associated (p value < 0 05 cluster corrected) with vitamin B12 concentrations, suggesting that elders with higher B12 levels had a more pronounced recruitment of this salience network region. Crucially, the activity of this area significantly predicted subjects' visual search and attention abilities (p value = 0 0023), whereas B12 levels per se failed to do so. Our results demonstrate that the relationship between blood biochemistry and elders' cognitive abilities is revealed when brain activity is included into the equation, thus highlighting the role of brain imaging as intermediate phenotype

CONFRONTO DEI PROFILI DI DISSOLUZIONE IN VITRO DI SPECIALITA’ MEDICINALI E FARMACI EQUIVALENTI CONTENENTI MESALAZINA

In questo lavoro, sono stati utilizzati due diversi metodi di dissoluzione in-vitro al fine di verificare come le diverse specialità medicinali e farmaci generici considerati fossero in grado di rilasciare il farmaco in condizioni temporali e di pH che simulano il passaggio delle compresse attraverso il tratto gastrointestinale; dallo stomaco al colon

Confronto dei profili di rilascio in vitro di formulazioni a rilascio ritardato, ph-dipendente, contenenti un principio attivo avente azione topica a livello della mucosa del colon

Scopo di questo lavoro è mettere a confronto i profili di rilascio di mesalazina da diverse formulazioni a rilascio ritardato, pH dipendente, presenti in commercio

Increased ex vivo expansion of late-endothelial progenitor cells in patients with Kaposi\u2019s sarcoma

Background Kaposi\u2019s Sarcoma (KS) is an angioproliferative disorder associated with human herpesvirus-8 (HHV-8) infection. Spindle cells are the predominant cell type in the KS lesions and are endothelial in origin. We previously demonstrated that late-Endothelial Progenitor Cells (late-EPCs) cultured ex vivo from KS patients are HHV-8-infected and support viral productive replication. Aim of this study was to investigate whether HHV-8 infection induced a different in vitro behaviour of KS late-EPCs. Methods Late-EPCs were cultured from peripheral blood mononuclear cells (PBMCs) of 15 classic KS patients and 15 healthy controls. Cultures from all subjects were observed for initial late-EPCs colony appearance, number of colonies, cell proliferation, cytokine production and in vitro angiogenesis. Results The average time of initial late-EPCs colony appearance was 19.42 \ub1 1.05 days in healthy controls and 12.84 \ub1 0.47 days in KS patients (P<0.001). The number of late-EPCs colonies/20x106 seeded PBMCs was 0.58 \ub1 0.08 in healthy controls and 5.64 \ub1 2.48 in KS patients (P<0.05). In this preliminary set of data no difference was observed in time of late-EPCs appearance and colony count related to clinical stage and evolution of KS disease. Conclusions We conclude that ex vivo expansion of late-EPCs is strikingly higher in KS patients than controls. These findings may be particularly relevant to KS pathogenesis, as late-EPCs are possibly considered putative precursors of spindle cells of KS lesion

Gemini Pyridinium Surfactants: Synthesis and Conductimetric Study of a Novel Class of Amphiphiles

A new series of pyridinium cationic gemini surfactants was prepared by quaternization of the 2,2'- (a,w alkanediyl)bispyridines with N-alkylating agents, whose reactivity is briefly discussed. Particularly useful was the use of long-chain alkyl triflates (trifluoromethanesulfonates) for both overcoming the sterical hindrance in the pyridines and obtaining higher synthetic yields. Wellknown 4,4' (a,w-alkanediyl)bis(1-alkylpyridinium) structures showed narrow temperature ranges for practical applications, due to their high Krafft points, while the new 2,2'-(a,w-alkanediyl)bis- (1-alkylpyridinium) series, accounted for good surface active properties. Due to the Krafft points below 0 °C, they could be exploited as solutions in water at any temperature. The characterization of the behavior of the series was performed by conductivity measurements. Some of the proposed structures exhibited unusual surface active behavior, which was interpreted in terms of particular conformational arrangements