Pasquale Spataro

Oxidative Protein Damage and Degradation in Lymphocytes from Patients Infected with Human Immunodeficiency Virus

It has been proposed that oxidative stress is the common mediator of apoptotic cell death in AIDS. However, mechanistic relationships between oxidative damage and cell death are far from clear. It is reported here that the mitogenic activation of T lymphocytes from human immunodeficiency virus-positive subjects involves perturbation of redox balance, as indicated by the increase in hydroethydine intracellular oxidation and manganese superoxide dismutase adaptive induction. Principal molecular targets of oxidative injury are cellular proteins whose content in carbonyl groups increases together with a dramatic increase in degradation of newly synthesized proteins catalyzed by the ATP- and ubiquitin-dependent proteolytic system. The major consequence of this metabolic anomaly is the decrease in protein cell mass leading to cells that are smaller than normal at lethal mitosis.

Multigenerational mitochondrial alterations in pneumocytes exposed to oil fly ash metals.

Oil fly ash (OFA), containing high amounts of transition metals, is among the most reactive airborne particulate matter emissions, which have been associated with several diseases, such as chronic obstructive pulmonary diseases (COPD), lung cancer, and cardiovascular diseases. The aim of the present study was to evaluate mitochondrial alterations in OFA-exposed cultured pneumocytes and in their progeny. Alveolar epithelial cells (A549 line) were exposed either to an OFA water solution, containing 68.8 μM vanadium (V), 110.4 μM iron (Fe), and 18.0 μM nickel (Ni), or to the individual metal solutions. Structural and functional mitochondrial parameters were determined in exposed cultures and in 3 consecutive subcultures. OFA, V and Fe solutions caused a time-dependent loss of mitochondrial enzymatic activity, glutathione depletion, generation of lipid hydroperoxides, hydrogen peroxide and other reactive oxygen species, especially in G(0)-G(1) phase cells, accompanied by a decrease in mitochondrial mass and transmembrane potential. Mitochondrial alterations were partly transmissible to daughter cells for up to 3 generations. Fe and especially V were responsible for the observed mitochondrial alterations in pneumocytes exposed to OFA. Spread of mitochondrial dysfunctions to daughter cells is expected to amplify oxidative stress in the respiratory epithelium and to play an important role in the pathogenesis of respiratory diseases.

Higher levels of oxidative DNA damage in cervical cells are correlated with the grade of dysplasia and HPV infection

The Human papillomavirus is responsible for the most common sexually transmitted infection and is also known to be an oncogenic virus that is associated with cervical, anogenital, and head‐neck cancers. The present study aims to assess whether oxidative DNA damage is correlated with the grade of HPV‐related lesions. Moreover, we evaluated clinical data and unhealthy lifestyles to verify their possible influence on the genesis of oxidative DNA damage in cervical cells. We quantified the amount of 8‐Oxo‐2′‐deoxyguanosine in DNA as a biomarker of oxidative damage in women with and without HPV infection. We also correlated oxidative damage with different stages of cervical lesions and available clinical data (e.g., HPV genotypes). To identify HPV infections, in which proteins with a transforming potential are produced, we performed a qualitative detection of HPV E6/E7 mRNA. Our results showed greater oxidative damage in HPV‐related dysplastic cervical lesions compared to samples with normal cytology, especially in women with high‐grade squamous intraepithelial lesions. The latter showed a closed link with high‐risk HPV genotypes. Reactive oxygen species can induce DNA double‐strand breaks in both the host DNA and in the circular viral episome; this could facilitate the integration of the virus, promoting HPV carcinogenesis. Therefore, in HPV‐infected women, it could be useful to reduce additional resources of reactive oxygen/nitrogen species (RONS) with a healthy lifestyle. J. Med. Virol. 88:336–344, 2016.

Mitochondrial dysfunction by pro-oxidant vanadium: Ex vivo assessment of individual susceptibility

The aim was to assess the individual susceptibility to mitochondrial impairment induced by ex vivo exposure to vanadium, an airborne pro-oxidant pollutant. In lymphocyte cultures V(IV)-treated of forty-five healthy subjects, we evaluated the mitochondrial transmembrane potential (Δψm) and the H2O2 in comparison to background values. As variables, we included both lifestyle factors and genetic polymorphisms (GSTM1 and GSTT1 variants, and C677T and A1298C variants of methylenetetrahydrofolate reductase MTHFR). H2O2 mitochondrial content increased significantly (P<0.05) after metal exposure while, in comparison to basal Δψm, both depolarisation and hyperpolarisation were recorded. This underlined the mitochondrial dysfunction vanadium-induced that worsens the redox imbalance by endogenous ROS overproduction. Only age was found to contribute significantly to the high inter-individual variability, as assessed by multivariate analysis. In older subjects, the H2O2/Δψm values underline the organelle impairment and, under V-exposure, Δψm values were inversely related to age (R=-0.591; P=0.012).

Intracellular accumulation of cell cycle regulatory proteins and nucleolin re-localization are associated with pre-lethal ultrastructural lesions in circulating T lymphocytes: The HIV-induced cell cycle dysregulation revisited

The HIV-induced demise of CD4-T cells is thought to be a result of the execution of genetically programmed cell death that occurs in lymphoid tissue, where many resident T cells are chronically hyperactivated. Since HIV-induced alterations of cell cycle control has been often indicated as prominent mechanism of immune hyper activation and cause of apoptotic death, the signal pathway involved in cell cycle dysregulation of T lymphocytes from HIV infected patients was extensively studied. Here, we also demonstrate that circulating T lymphocytes leave lymphoid tissues with diffused regressive lesions (vacuolization, blebbing, nuclear evanescence and organelle swelling). Equally diffused are biochemical anomalies that accompany the overall disarrangement of cell structure, particularly the fragmentation and diffusion into the cytoplasm of C23/nucleolin, the intracellular accumulation of short lived regulatory proteins and the decrease in expression of membrane proteins. All this is something more than a cell cycle-related remodelling of cell morphology and biochemical mechanisms, and rather recalls a necrotic/oncotic cell damage. Since these changes are associated with adaptive mechanisms to hypoxia, we give evidence for alteration of cell cycle control developing in conditions of scarce energy supply.

Prevalence of SENV-H and SENV-D Virus: Epidemiological Study in Blood Donors and Dialysis Patients

Introduction. Recently, the identification of the SEN virus as a possible etiological agent of parental transmission hepatitis led to the study of the prevalence of such pathogen agents, particularly SENV-H, in our population. This paper compares the rate prevalence in high-risk subjects, such as dialysis patients, and low-risk subjects, such as blood donors. Material and Methods. The study was carried out on SEN virus DNA extracted from serum of dialysis patients and blood donors, and the presence of viral genomes was performed by the nested PCR method. Results. The results showed a higher prevalence in male blood donors, supporting the hypothesis of an epidemiological role for sexual and also parental transmission, as is clearly demonstrated by the high prevalence in dialysis patients. The result reduced the importance of the possible etiological role of the SEN virus due to the high percentage of positivity in healthy population, and it induces one to consider poorly significant the pathogenicity of such viral agents. Conclusion. For this instance, the authors, in agreement with the phylogenically related TT virus, described SEN viruses as absolutely not pathogens and considered them as “simple guests.”

HIV genomic mutations causing resistance to antiretroviral drugs in seropositive Sicilians.

Through the use of highly active antiretroviral therapy a significant reduction occurred in mortality and morbidity caused by Human Immunodeficiency Virus. The use of antiretroviral drugs resulted in the emergence of resistant viral strains due to mutations that cause a selective advantage to the virus. The aim of our study is to monitor the HIV-1 infection in Sicilians patients evaluating the presence of mutations that make the virus resistant to the therapy. The QIAGEN QIAamp Viral RNA Mini Kit was used to extract HIV-1 viral RNA from 300 patients while the TRUGENE HIV-1 Genotyping Kit and the OpenGene DNA Sequencing System determined viral mutations in the RNA samples. The analysis showed that from 300 subjects, 116 developed Antiretroviral Drug Resistance. The percentage of patients with resistance to nucleoside reverse transcriptase inhibitor (NRTI), non nucleoside reverse transcriptase inhibitor (NNRTI) and protease inhibitor was 26%, 23% and 20%, respectively. Comparison between drug resistances and mutations showed that 134 individuals had mutations in genes codifying for reverse transcriptase but a little more than 50% were associated with resistance to reverse transcriptase inhibitors, in particular 78 and 68 subjects developed drug resistances to NRTI and NNRTI classes respectively. Subjects that showed mutations in genes codifying for protease were 216 but only 59 of these were associated with resistance to protease inhibitors. Our findings emphasize the importance of continued resistance surveillance. Monitoring of transmitted resistance continues to be needed among treatment-exposed patients because of the benefit it provides for the development of drugs effective against the most frequently found drug-resistant viruses.

Nucleolin relocalization associated with pre-lethal alterations of T cell morphology: redefining cell death in HIV infection

Results Here we demonstrate that circulating T lymphocytes, both CD4+ and CD8+, leave lymphoid tissues with diffused regressive lesions such as vacuolization, blebbing, nuclear evanescence and organelle swelling. Equally diffused are biochemical anomalies that accompany the overall disarrangement of cell structure, namely (i) fragmentation and diffusion into the cytoplasm of C23/ nucleolin, the principal structural protein of the nucleus (ii) an accumulation of short lived regulatory proteins (p16, p21 and p53), likely due to the progressive extinction of the ATP ub proteasome system and (iii) a decreased expression of membrane proteins.

Infezione da SENV D e SENV - H in soggetti sani ed in pazienti emodializzati

Obiettivi a) valutare la sovrapponibilita dei risultati con i dati nazionali e internazionali ricercando il SENV-D e il SENV-H in un campione di popolazione sana, costituita da donatori di sangue di Messina; b) valutare se la presenza di tali virus, in soggetti dializzati di tre centri di dialisi locali, sia significativa rispetto alla popolazione sana e se la prevalenza sia sovrapponibile a quella riportata in letteratura; c) fornire, visti i pochi dati esistenti in bibliografia riguardo la diffusione del SENV nei soggetti dializzati, un elemento epidemiologico inesistente attualmente nella nostra zona. d) adottare, eventualmente, misure preventive nei sopraccitati centri di dialisi Metodo: la presenza del SENV-D e SENV-H e effettuata mediante metodica PCR: Il DNA viene estratto dai campioni con il kit QIAamp DNA e gli amplificati sono rivelati tramite elettroforesi su gel di agarosio. Risultati : e stata evidenziata, sui 50 donatori di sangue sin qui testati, una percentuale di positivita per SENV - H del 2% e dello 0% per SENV - D. Per quanto riguarda i 20 soggetti dializzati finora esaminati ed appartenenti ad uno dei sopracitati centri, e stata riscontrata una positivita solo per SENV – H pari al 10%. Conclusioni : i risultati preliminarmente ottenuti sulla popolazione sana, evidenziano una sovrapponibilita di diffusione del SENV – H con i dati riportati da altri autori in letteratura, mentre quelli relativi ai dializzati rivelano una positivita piu bassa.