HIV Infection, Antiretroviral Therapy and Cardiovascular Risk

In the last 15 years, highly active antiretroviral therapy (HAART) has determined a dramatic reduction of both morbidity and mortality in human immunodeficiency virus (HIV)-infected subjects, transforming this infection in a chronic and manageable disease. Patients surviving with HIV in the developed world, in larger number men, are becoming aged. As it would be expected for a population of comparable age, many HIV-infected individuals report a family history of cardiovascular disease, a small proportion have already experienced a cardiovascular event and an increasing proportion has diabetes mellitus. Smoking rate is very high while an increasing proportion of HIV-infected individuals have dyslipidaemia. Studies suggest that these traditional risk factors could play an important role in the development of cardiovascular disease in these patients as they do in the general population. Thus, whilst the predicted 10-year cardiovascular disease risk remains relatively low at present, it will likely increase in relation to the progressive aging of this patient population. Thus, the long-term follow-up of HIV infected patients has to include co-morbidity management such as cardiovascular disease prevention and treatment. Two intriguing aspects related to the cardiovascular risk in patients with HIV infection are the matter of current investigation: 1) while these subjects share many cardiovascular risk factors with the general population, HIV infection itself increases cardiovascular risk; 2) some HAART regimens too influence atherosclerotic profile, partly due to lipid changes. Although the mechanisms involved in the development of cardiovascular complications in HIV-infected patients remain to be fully elucidated, treatment guidelines recommending interventions to prevent cardiovascular disease in these individuals are already available; however, their application is still limited

Freeze-thawing of liposomes: investigation of cryoprotectants for freeze-drying

Liposomes are phospholipid membranes widely used for drug delivery in biomedical applications. They can retain, protect and deliver macromolecules to target tissues and then control the release of their cargoes. To exploit this function, the liposomal membrane integrity is crucial to avoid undesired leakage of cargoes. In this work, the influence of low temperature storing conditions on the stability of liposomes is investigated for further delivery applications

Cytotoxicity and Thermal Characterization Assessment of Excipients for the Development of Innovative Lyophilized Formulations for Oncological Applications

Freeze-drying, also known as lyophilization, significantly improves the storage, stability, shelf life, and clinical translation of biopharmaceuticals. On the downside, this process faces complex challenges, i.e., the presence of freezing and drying stresses for the active compounds, the uniformity and consistency of the final products, and the efficiency and safety of the reconstituted lyophilized formulations. All these requirements can be addressed by adding specific excipients that can protect and stabilize the active ingredient during lyophilization, assisting in the formation of solid structures without interfering with the biological and/or pharmaceutical action of the reconstituted products. However, these excipients, generally considered safe and inert, could play an active role in the formulation interacting with the biological cellular machinery and promoting toxicity. Any side effects should be carefully identified and characterized to better tune any treatments in terms of concentrations and administration times. In this work, various concentrations in the range of 1 to 100 mg/mL of cellobiose, lactose, sucrose, trehalose, isoleucine, glycine, methionine, dextran, mannitol, and (2-hydroxypropyl)-β-cyclodextrin were evaluated in terms of their ability to create uniform and solid lyophilized structures. The freeze-dried products were then reconstituted in the appropriate cell culture media to assess their in vitro cytotoxicity on both a healthy cell line (B-lymphocytes) and their tumoral lymphoid counterpart (Daudi). Results showed that at 10 mg/mL, all the excipients demonstrated suitable lyophilized solid structures and high tolerability by both cell lines, while dextran was the only excipient well-tolerated also up to 100 mg/mL. An interesting result was shown for methionine, which even at 10 mg/mL, selectively affected the viability of the cancerous cell line only, opening future perspectives for antitumoral applications

Comparative Studies of Different Preservation Methods and Relative Freeze-Drying Formulations for Extracellular Vesicle Pharmaceutical Applications

Extracellular vesicles (EVs) have been studied for years for their role as effectors and mediators of cell-to-cell communication and their potential application to develop new and increasingly performing nanotechnological systems for the diagnosis and/or treatment of many diseases. Given all the EVs applications as just isolated, functionalized, or even engineered cellular-derived pharmaceuticals, the standardization of reliable and reproducible methods for their preservation is urgently needed. In this study, we isolated EVs from a healthy blood cell line, B lymphocytes, and compared the effectiveness of different storage methods and relative freeze-drying formulations to preserve some of the most important EVs’ key features, i.e., concentration, mean size, protein content, and surface antigen’s expression. To develop a preservation method that minimally affects the EVs’ integrity and functionality, we applied the freeze-drying process in combination with different excipients. Since EVs are isolated not only from body fluids but also from culture media conditioned by the cells growing there, we decided to test both the effects of the traditional pharmaceutical excipient and of biological media to develop EVs solidified products with desirable appearance and performance properties. Results showed that some of the tested excipients, i.e., sugars in combination with dextran and glycine, successfully maintained the stability and integrity of EVs upon lyophilization. In addition, to evaluate the preservation of the EVs’ biological activity, we assessed the cytotoxicity and internalization ability of the reconstituted EVs in healthy (B lymphocytes) and tumoral (Burkitt’s lymphoma) cells. Reconstituted EVs demonstrated toxicity only toward the cancerous cells, opening new therapeutic opportunities for the oncological field. Furthermore, our study showed how some biological or cellular-conditioned fluids, commonly used in the field of cell cultures, can act not only as cryoprotectants but also as active pharmaceutical ingredients, significantly tuning the therapeutic effect of EVs, even increasing their cellular internalization

Facile Chemical Synthesis of Doped ZnO Nanocrystals Exploiting Oleic Acid

Zinc oxide nanocrystals (ZnO-NCs) doped with transition metal elements or rare earth elements can be probed for magnetic resonance imaging to be used as a molecular imaging technique for accurate diagnosis of various diseases. Herein, we use Mn as a candidate of transition metal elements and Gd as a presenter of rare earth elements. We report an easy and fast coprecipitation method exploiting oleic acid to synthesize spherical-shaped, small-sized doped ZnO-NCs. We show the improved colloidal stability of oleate-stabilized doped ZnO-NCs compared to the doped ZnO-NCs synthesized by conventional sol-gel synthesis method, i.e., without a stabilizing agent, especially for the Mn dopant. We also analyze their structural, morphological, optical, and magnetic properties. We are able to characterize the persistence of the crystalline properties (wurtzite structure) of ZnO in the doped structure and exclude the formation of undesired oxides by doping elements. Importantly, we determine the room-temperature ferromagnetism of the doped ZnO-NCs. This oleate-stabilized coprecipitation method can be subjected as a standard procedure to synthesize doped and also co-doped ZnO-NCs with any transition metal elements or rare earth elements. In the future, oleate-stabilized Gd/Mn-doped ZnO-NCs can be exploited as magnetic resonance imaging (MRI) contrast agents and possibly increase the signal intensity on T1-weighted images or reduce the signal intensity on T2-weighted images

Partial protective effect of CCR5-Delta 32 heterozygosity in a cohort of heterosexual Italian HIV-1 exposed uninfected individuals

Despite multiple sexual exposure to HIV-1 virus, some individuals remain HIV-1 seronegative (exposed seronegative, ESN). The mechanisms underlying this resistance remain still unclear, although a multifactorial pathogenesis can be hypothesised. Although several genetic factors have been related to HIV-1 resistance, the homozigosity for a mutation in CCR5 gene (the 32 bp deletion, i.e. CCR5-Delta32 allele) is presently considered the most relevant one. In the present study we analysed the genotype at CCR5 locus of 30 Italian ESN individuals (case group) who referred multiple unprotected heterosexual intercourse with HIV-1 seropositive partner(s), for at least two years. One hundred and twenty HIV-1 infected patients and 120 individuals representative of the general population were included as control groups. Twenty percent of ESN individuals had heterozygous CCR5-Delta 32 genotype, compared to 7.5% of HIV-1 seropositive and 10% of individuals from the general population, respectively. None of the analysed individuals had CCR5-Delta 32 homozygous genotype. Sequence analysis of the entire open reading frame of CCR5 was performed in all ESN subjects and no polymorphisms or mutations were identified. Moreover, we determined the distribution of C77G variant in CD45 gene, which has been previously related to HIV-1 infection susceptibility. The frequency of the C77G variant showed no significant difference between ESN subjects and the two control groups. In conclusion, our data show a significantly higher frequency of CCR5-Delta 32 heterozygous genotype (p = 0.04) among the Italian heterosexual ESN individuals compared to HIV-1 seropositive patients, suggesting a partial protective role of CCR5-Delta 32 heterozygosity in this cohort

Assessment of seasonal influenza vaccine effectiveness in patients from a central Italy reference hospital: pitfalls and intricacies from a pilot case-control study

Objectives: Influenza vaccination protects high-risk populations from severe outcomes. We assessed the feasibility of testing influenza vaccine effectiveness against hospitalization with laboratory-confirmed influenza. Methods: All hospitalized patients with influenza-like illness within 14 days, were swabbed. Cases were positive at RT-PCR for influenza A/B. Results: AtRome “GemelliHospital” (Season 2011-2012) 104 patients were contacted and 62 recruited. Considering total sample and target group (n= 47, 76%), only 29% and 38% had been vaccinated. Eighteen patients were laboratory-confirmed for influenza. Conclusions: RecruitedILI patients and prevalence of vaccinated subjects were less than expected. Larger numbers are warranted to study vaccine effectiveness against severe influenza outcomes. &nbsp