Effect of Cigarette Smoking on Clinical and Molecular Endpoints in COPD Patients.

Cigarette smoking is a primary contributor to mortality risks and is associated with various diseases. Among these, COPD represents a significant contributor to global mortality and disability. The objective of this study is to investigate the effect of smoking on a selected battery of variables, with an emphasis on DNA damage. A total of 87 elderly patients diagnosed with COPD, divided into three groups based on their smoking history (current, former, never-smokers), were evaluated using a cross-sectional approach. Clinical features including mortality and inflammatory/oxidative parameters (Lymphocytes/Monocytes, Neutrophils/Lymphocytes, Platelets/Lymphocytes ratio), SII, MDA, 8-Oxo-dG, and IL6 (ELISA assay), as well as DNA damage (comet assay), were investigated. Virus infection, i.e., influenza A virus subtype H1N1, JC polyomavirus (JCPyV), BK polyomavirus (BKPyV), and Torquetenovirus (TTV), was also tested. Current smokers exhibit higher levels of comorbidity (CIRS; p < 0.001), Platelets/Lymphocytes ratio (p < 0.001), systemic immune inflammation (p < 0.05), and DNA damage (p < 0.001). Former smokers also showed higher values for parameters associated with oxidative damage and showed a much lower probability of surviving over 5 years compared to never- and current smokers (p < 0.0017). This study showed a clear interaction between events which are relevant to the oxidative pathway and cigarette smoking. A category of particular interest is represented by former smokers, especially for lower survival, possibly due to the presence of more health problems. Our findings raise also the attention to other parameters which are significantly affected by smoking and are useful to monitor COPD patients starting a program of pulmonary rehabilitation (DNA damage, inflammation parameters, and selected viral infections).

Correlation between Chest Computed Tomography Score and Laboratory Biomarkers in the Risk Stratification of COVID-19 Patients Admitted to the Emergency Department.

BACKGROUND: It has been reported that mid-regional proadrenomedullin (MR-proADM) could be considered a useful tool to stratify the mortality risk in COVID-19 patients upon admission to the emergency department (ED). During the COVID-19 outbreak, computed tomography (CT) scans were widely used for their excellent sensitivity in diagnosing pneumonia associated with SARS-CoV-2 infection. However, the possible role of CT score in the risk stratification of COVID-19 patients upon admission to the ED is still unclear. AIM: The main objective of this study was to assess if the association of the CT findings alone or together with MR-proADM results could ameliorate the prediction of in-hospital mortality of COVID-19 patients at the triage. Moreover, the hypothesis that CT score and MR-proADM levels together could play a key role in predicting the correct clinical setting for these patients was also evaluated. METHODS: Epidemiological, demographic, clinical, laboratory, and outcome data were assessed and analyzed from 265 consecutive patients admitted to the triage of the ED with a SARS-CoV-2 infection. RESULTS AND CONCLUSIONS: The accuracy results by AUROC analysis and statistical analysis demonstrated that CT score is particularly effective, when utilized together with the MR-proADM level, in the risk stratification of COVID-19 patients admitted to the ED, thus helping the decision-making process of emergency physicians and optimizing the hospital resources.

Laboratory Automation in Microbiology: Impact on Turnaround Time of Microbiological Samples in COVID Time.

BACKGROUND: Laboratory Automation (LA) is an innovative technology that is currently available for microbiology laboratories. LA can be a game changer by revolutionizing laboratory workflows through efficiency improvement and is also effective in the organization and standardization of procedures, enabling staff requalification. It can provide an important return on investment (time spent redefining the workflow as well as direct costs of instrumentation) in the medium to long term. METHODS: Here, we present our experience with the WASPLab® system introduced in our lab during the COVID-19 pandemic. We evaluated the impact due to the system by comparing the TAT recorded on our samples before, during, and after LA introduction (from 2019 to 2021). We focused our attention on blood cultures (BCs) and biological fluid samples (BLs). RESULTS: TAT recorded over time showed a significant decrease: from 97 h to 53.5 h (Δ43.5 h) for BCs and from 73 h to 58 h (Δ20 h) for BLs. Despite the introduction of the WASPLab® system, we have not been able to reduce the number of technical personnel units dedicated to the microbiology lab, but WASPLab® has allowed us to direct some of the staff resources toward other laboratory activities, including those required by the pandemic. CONCLUSIONS: LA can significantly enhance laboratory performance and, due to the significant reduction in reporting time, can have an effective impact on clinical choices and therefore on patient outcomes. Therefore, the initial costs of LA adoption must be considered worthwhile.

Influenza virus replication is affected by glutaredoxin1-mediated protein deglutathionylation.

Several redox modifications have been described during viral infection, including influenza virus infection, but little is known about glutathionylation and this respiratory virus. Glutathionylation is a reversible, post-translational modification, in which protein cysteine forms transient disulfides with glutathione (GSH), catalyzed by cellular oxidoreductases and in particular by glutaredoxin (Grx). We show here that (i) influenza virus infection induces protein glutathionylation, including that of viral proteins such as hemagglutinin (HA); (ii) Grx1-mediated deglutathionylation is important for the viral life cycle, as its inhibition, either with an inhibitor of its enzymatic activity or by siRNA, decreases viral replication. Overall these data contribute to the characterization of the complex picture of redox regulation of the influenza virus replication cycle and could help to identify new targets to control respiratory viral infection.

COVID-19 as a Potential Cause of Muscle Injuries in Professional Italian Serie A Soccer Players: A Retrospective Observational Study.

The COVID-19 pandemic has shocked the entire planet. The soccer world has also suffered major upheavals, and many professional soccer players have been infected with the virus. The aim of this study was to evaluate the incidence of injuries in Italian Serie A professional soccer players before and during the COVID-19 pandemic. Methods: We evaluated the incidence of muscle injuries between four competitive seasons of the Italian Serie A (2016-2017, 2017-2018, and 2018-2019 pre-COVID-19 vs. 2020/2021 post-COVID-19) in professional soccer players. Results: Significant differences were found in muscular injuries between the post-COVID-19 season and the previous seasons (p < 0.001). The median split of the players' positivity duration was of 15 days. The players' long positivity (PLP) group showed a significant number of muscular injuries compared to the players' short positivity (PSP) group (p < 0.0014, ES = 0.81, Large). The total teams' days of positivity were significantly related to the total team number of muscular injuries (r = 0.86; CI 95% 0.66 to 0.94; p < 0.0001). In conclusion, this data showed that the competitive season post-COVID-19 lockdown has a higher incidence of muscle injuries in Italian Serie A soccer players compared to the pre-pandemic competitive season.

COS-7 and SVGp12 Cellular Models to Study JCPyV Replication and MicroRNA Expression after Infection with Archetypal and Rearranged-NCCR Viral Strains.

Since the non-coding control region (NCCR) and microRNA (miRNA) could represent two different and independent modalities of regulating JC polyomavirus (JCPyV) replication at the transcriptional and post-transcriptional levels, the interplay between JC viral load based on NCCR architecture and miRNA levels, following JCPyV infection with archetypal and rearranged (rr)-NCCR JCPyV variants, was explored in COS-7 and SVGp12 cells infected by different JCPyV strains. Specifically, the involvement of JCPyV miRNA in regulating viral replication was investigated for the archetypal CY strain-which is the transmissible form-and for the rearranged MAD-1 strain, which is the first isolated variant from patients with progressive multifocal leukoencephalopathy. The JCPyV DNA viral load was low in cells infected with CY compared with that in MAD-1-infected cells. Productive viral replication was observed in both cell lines. The expression of JCPyV miRNAs was observed from 3 days after viral infection in both cell types, and miR-J1-5p expression was inversely correlated with the JCPyV replication trend. The JCPyV miRNAs in the exosomes present in the supernatants produced by the infected cells could be carried into uninfected cells. Additional investigations of the expression of JCPyV miRNAs and their presence in exosomes are necessary to shed light on their regulatory role during viral reactivation.

Adaptive strategies of uropathogenic Escherichia coli CFT073: from growth in lab media to virulence during host cell adhesion / Estrategias adaptativas de Escherichia coli CFT073 uropatógena: desde el crecimiento en medios de laboratorio hasta la virulencia durante la adhesión de la célula huésped

Urinary tract infections (UTIs) are a major concern in public health. The prevalent uropathogenic bacterium in healthcare settings is Escherichia coli. The increasing rate of antibiotic-resistant strains demands studies to understand E. coli pathogenesis to drive the development of new therapeutic approaches. This study compared the gene expression profile of selected target genes in the prototype uropathogenic E. coli (UPEC) strain CFT073 grown in Luria Bertani (LB), artificial urine (AU), and during adhesion to host bladder cells by semi-quantitative real-time PCR (RT-PCR) assays. AU effectively supported the growth of strain CFT073 as well as other E. coli strains with different lifestyles, thereby confirming the appropriateness of this medium for in vitro models. Unexpectedly, gene expression of strain CFT073 in LB and AU was quite similar; conversely, during the adhesion assay, adhesins and porins were upregulated, while key global regulators were downregulated with respect to lab media. Interestingly, fimH and papGII genes were significantly expressed in all tested conditions. Taken together, these results provide for the first time insights of the metabolic and pathogenic profile of strain CFT073 during the essential phase of host cell adhesion.(AU)

Long Lasting Cellular Immune Response Induced by mRNA Vaccination: Implication for Prevention Strategies.

As the COVID19 pandemic continues to spread and vaccinations are administered throughout the world at different rates and with different strategies, understanding the multiple aspects of the immune response to vaccinations is required to define more efficient vaccination strategies. To date, the duration of protection induced by COVID19 vaccines is still matter of debate. To assess whether 2-doses vaccination with BNT162b2 mRNA COVID-19 vaccine was sufficient to induce a persistent specific cellular immune response, we evaluated the presence of SARS-COV2 Spike-specific B and T lymphocytes in 28 healthcare workers 1 and 7 months after completing the vaccination cycle. The results showed that at 7 months after second dose a population of Spike-specific B lymphocytes was still present in 86% of the immunized subjects, with a higher frequency when compared to not-immunized controls (0.38% ± 0.07 vs 0.13% ± 0.03, p<0.001). Similarly, specific CD4+ and CD8+ T lymphocytes, able to respond in vitro to stimulation with Spike derived peptides, were found at 7 months. These results confirm that vaccination with BNT162b2 is able to induce a specific immune response, potentially long lasting, and could be helpful in defining future vaccination strategies.

Adaptive strategies of uropathogenic Escherichia coli CFT073: from growth in lab media to virulence during host cell adhesion.

Urinary tract infections (UTIs) are a major concern in public health. The prevalent uropathogenic bacterium in healthcare settings is Escherichia coli. The increasing rate of antibiotic-resistant strains demands studies to understand E. coli pathogenesis to drive the development of new therapeutic approaches. This study compared the gene expression profile of selected target genes in the prototype uropathogenic E. coli (UPEC) strain CFT073 grown in Luria Bertani (LB), artificial urine (AU), and during adhesion to host bladder cells by semi-quantitative real-time PCR (RT-PCR) assays. AU effectively supported the growth of strain CFT073 as well as other E. coli strains with different lifestyles, thereby confirming the appropriateness of this medium for in vitro models. Unexpectedly, gene expression of strain CFT073 in LB and AU was quite similar; conversely, during the adhesion assay, adhesins and porins were upregulated, while key global regulators were downregulated with respect to lab media. Interestingly, fimH and papGII genes were significantly expressed in all tested conditions. Taken together, these results provide for the first time insights of the metabolic and pathogenic profile of strain CFT073 during the essential phase of host cell adhesion.

Diagnostic Value of JC Polyomavirus Viruria, Viremia, Serostatus and microRNA Expression in Multiple Sclerosis Patients Undergoing Immunosuppressive Treatment.

Markers of JC polyomavirus (JCPyV) activity can be used to evaluate the risk of progressive multifocal leukoencephalopathy (PML) in treated multiple sclerosis (MS) patients. The presence of JCPyV DNA and microRNA (miR-J1-5p), the anti-JCV index and the sequence of the non-coding control region (NCCR) in urine and plasma were determined in 42 MS subjects before treatment (T0), 6 months (T6) and 12 months (T12) after natalizumab, ocrelizumab, fingolimod or dimethyl-fumarate administration and in 25 healthy controls (HC). The number of MS patients with viruria increased from 43% at T0 to 100% at T12, whereas it remained similar for the HC group (35-40%). Viremia first occurred 6 months after treatment in MS patients and increased after 12 months, whereas it was absent in HC. The viral load in urine and plasma from the MS cohort increased over time, mostly pronounced in natalizumab-treated patients, whereas it persisted in HC. The archetypal NCCR was detected in all positive urine, whereas mutations were observed in plasma-derived NCCRs resulting in a more neurotropic variant. The prevalence and miR-J1-5p copy number in MS urine and plasma dropped after treatment, whereas they remained similar in HC specimens. Viruria and miR-J1-5p expression did not correlate with anti-JCV index. In conclusion, analyzing JCPyV DNA and miR-J1-5p levels may allow monitoring JCPyV activity and predicting MS patients at risk of developing PML.

BK Polyomavirus Activates HSF1 Stimulating Human Kidney Hek293 Cell Proliferation.

OBJECTIVES: Some DNA viruses, such as BKPyV, are capable of inducing neoplastic transformation in human tissues through still unclear mechanisms. The goal of this study is to investigate the carcinogenic potential of BK polyomavirus (BKPyV) in human embryonic kidney 293 (Hek293) cells, dissecting the molecular mechanism that determines the neoplastic transformation. MATERIALS AND METHODS: BKPyV, isolated from urine samples of infected patients, was used to infect monolayers of Hek293 cells. Subsequently, intracellular redox changes, GSH/GSSH concentration by HPLC, and reactive oxygen/nitrogen species (ROS/RNS) production were monitored. Moreover, to understand the signaling pathway underlying the neoplastic transformation, the redox-sensitive HFS1-Hsp27 molecular axis was examined using the flavonoid quercetin and polishort hairpin RNA technologies. RESULTS: The data obtained show that while BKPyV replication is closely linked to the transcription factor p53, the increase in Hek293 cell proliferation is due to the activation of the signaling pathway mediated by HSF1-Hsp27. In fact, its inhibition blocks viral replication and cell growth, respectively. CONCLUSIONS: The HSF1-Hsp27 signaling pathway is involved in BKPyV infection and cellular replication and its activation, which could be involved in cell transformation.

Risk Assessment of Progressive Multifocal Leukoencephalopathy in Multiple Sclerosis Patients during 1 Year of Ocrelizumab Treatment.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) caused by the JC virus is the main limitation to the use of disease modifying therapies for treatment of multiple sclerosis (MS). METHODS: To assess the PML risk in course of ocrelizumab, urine and blood samples were collected from 42 MS patients at baseline (T0), at 6 (T2) and 12 months (T4) from the beginning of therapy. After JCPyV-DNA extraction, a quantitative-PCR (Q-PCR) was performed. Moreover, assessment of JCV-serostatus was obtained and arrangements' analysis of non-coding control region (NCCR) and of viral capsid protein 1 (VP1) was carried out. RESULTS: Q-PCR revealed JCPyV-DNA in urine at all selected time points, while JCPyV-DNA was detected in plasma at T4. From T0 to T4, JC viral load in urine was detected, increased in two logarithms and, significantly higher, compared to viremia. NCCR from urine was archetypal. Plasmatic NCCR displayed deletion, duplication, and point mutations. VP1 showed the S269F substitution involving the receptor-binding region. Anti-JCV index and IgM titer were found to statistically decrease during ocrelizumab treatment. CONCLUSIONS: Ocrelizumab in JCPyV-DNA positive patients is safe and did not determine PML cases. Combined monitoring of ocrelizumab's effects on JCPyV pathogenicity and on host immunity might offer a complete insight towards predicting PML risk.

SARS-CoV-2: Comparative analysis of different RNA extraction methods.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. Currently, several automatic methods that include RNA extraction are available. However, due to the growing demand, a shortage in kit supplies could be experienced in several labs. For these reasons, the use of different commercial or in-house protocols for RNA extraction may increase the possibility to analyze high number of samples. Herein, we compared the efficiency of RNA extraction of three different commercial kits and an in-house extraction protocol using synthetic ssRNA standards of SARS-CoV-2 as well as in oro-nasopharyngeal swabs from six COVID-19-positive patients. It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection.

Glutathione and Nitric Oxide: Key Team Players in Use and Disuse of Skeletal Muscle.

Glutathione (GSH) is the main non-enzymatic antioxidant playing an important role in detoxification, signal transduction by modulation of protein thiols redox status and direct scavenging of radicals. The latter function is not only performed against reactive oxygen species (ROS) but GSH also has a fundamental role in buffering nitric oxide (NO), a physiologically-produced molecule having-multifaceted functions. The efficient rate of GSH synthesis and high levels of GSH-dependent enzymes are characteristic features of healthy skeletal muscle where, besides the canonical functions, it is also involved in muscle contraction regulation. Moreover, NO production in skeletal muscle is a direct consequence of contractile activity and influences several metabolic myocyte pathways under both physiological and pathological conditions. In this review, we will consider the homeostasis and intersection of GSH with NO and then we will restrict the discussion on their role in processes related to skeletal muscle function and degeneration.

Hemodialysis biomarkers: total advanced glycation end products (AGEs) against oxidized human serum albumin (HSAox).

AIMS: Nephropathic patients show higher levels of advanced glycation end products (AGEs) and oxidized human serum albumin (HSAox) compared to healthy subjects. These two classes of compounds are formed as the result of oxidative insults; for this reason, they can be useful oxidative stress biomarkers. The present study examines the variation of AGEs and HSAox in hemodialysis (HD) patients before and after dialysis session, evaluating the impact of different dialytic techniques and filters on their removal. METHODS: A total of 50 healthy subjects (control group) and 130 HD patients were enrolled in the study. Hemodialysis patients were subdivided based on dialytic techniques: 109 in diffusive technique and 22 in convective technique. We monitored HSAox, AGEs and other laboratory parameters at early morning in healthy subjects and in HD patients before and after the dialysis procedures. RESULTS: The level of HSAox decreases after a single dialytic session (from 58.5 ± 8.8% to 41.5 ± 11.1%), but the concentration of total AGEs increases regardless of adopted dialytic techniques (from 6.8 ± 5.2 µg/ml to 9.2 ± 4.4 µg/ml). In our study, levels of HSAox and total AGEs are similar in diabetic and non-diabetic HD patients. The increase in total AGEs after dialysis was only observed using polysulfone filters but was absent with polymethacrylate filters. CONCLUSIONS: HSAox is a simple and immediate method to verify the beneficial effect of a single dialysis session on the redox imbalance, always present in HD patients. Total AGEs assayed by ELISA procedure seem to be a less reliable biomarker in this population.

Role of Glutathionylation in Infection and Inflammation.

Glutathionylation, that is, the formation of mixed disulfides between protein cysteines and glutathione (GSH) cysteines, is a reversible post-translational modification catalyzed by different cellular oxidoreductases, by which the redox state of the cell modulates protein function. So far, most studies on the identification of glutathionylated proteins have focused on cellular proteins, including proteins involved in host response to infection, but there is a growing number of reports showing that microbial proteins also undergo glutathionylation, with modification of their characteristics and functions. In the present review, we highlight the signaling role of GSH through glutathionylation, particularly focusing on microbial (viral and bacterial) glutathionylated proteins (GSSPs) and host GSSPs involved in the immune/inflammatory response to infection; moreover, we discuss the biological role of the process in microbial infections and related host responses.

Corrigendum: GSH-C4 Acts as Anti-inflammatory Drug in Different Models of Canonical and Cell Autonomous Inflammation Through NFκB Inhibition.

[This corrects the article DOI: 10.3389/fimmu.2019.00155.].

Mitophagy and Oxidative Stress in Cancer and Aging: Focus on Sirtuins and Nanomaterials.

Mitochondria are the cellular center of energy production and of several important metabolic processes. Mitochondrion health is maintained with a substantial intervention of mitophagy, a process of macroautophagy that degrades selectively dysfunctional and irreversibly damaged organelles. Because of its crucial duty, alteration in mitophagy can cause functional and structural adjustment in the mitochondria, changes in energy production, loss of cellular adaptation, and cell death. In this review, we discuss the dual role that mitophagy plays in cancer and age-related pathologies, as a consequence of oxidative stress, evidencing the triggering stimuli and mechanisms and suggesting the molecular targets for its therapeutic control. Finally, a section has been dedicated to the interplay between mitophagy and therapies using nanoparticles that are the new frontier for a direct and less invasive strategy.

Recurrent herpes simplex virus-1 infection induces hallmarks of neurodegeneration and cognitive deficits in mice.

Herpes simplex virus type 1 (HSV-1) is a DNA neurotropic virus, usually establishing latent infections in the trigeminal ganglia followed by periodic reactivations. Although numerous findings suggested potential links between HSV-1 and Alzheimer's disease (AD), a causal relation has not been demonstrated yet. Hence, we set up a model of recurrent HSV-1 infection in mice undergoing repeated cycles of viral reactivation. By virological and molecular analyses we found: i) HSV-1 spreading and replication in different brain regions after thermal stress-induced virus reactivations; ii) accumulation of AD hallmarks including amyloid-ß protein, tau hyperphosphorylation, and neuroinflammation markers (astrogliosis, IL-1ß and IL-6). Remarkably, the progressive accumulation of AD molecular biomarkers in neocortex and hippocampus of HSV-1 infected mice, triggered by repeated virus reactivations, correlated with increasing cognitive deficits becoming irreversible after seven cycles of reactivation. Collectively, our findings provide evidence that mild and recurrent HSV-1 infections in the central nervous system produce an AD-like phenotype and suggest that they are a risk factor for AD.

GSH-C4 Acts as Anti-inflammatory Drug in Different Models of Canonical and Cell Autonomous Inflammation Through NFκB Inhibition.

An imbalance in GSH/GSSG ratio represents a triggering event in pro-inflammatory cytokine production and inflammatory response. However, the molecular mechanism(s) through which GSH regulates macrophage and cell autonomous inflammation remains not deeply understood. Here, we investigated the effects of a derivative of GSH, the N-butanoyl glutathione (GSH-C4), a cell permeable compound, on lipopolisaccharide (LPS)-stimulated murine RAW 264.7 macrophages, and human macrophages. LPS alone induces a significant production of pro-inflammatory cytokines, such as IL-1ß, IL-6, and TNF-α and a significant decrement of GSH content. Such events were significantly abrogated by treatment with GSH-C4. Moreover, GSH-C4 was highly efficient in buffering cell autonomous inflammatory status of aged C2C12 myotubes and 3T3-L1 adipocytes by suppressing the production of pro-inflammatory cytokines. We found that inflammation was paralleled by a strong induction of the phosphorylated form of NFκB, which translocates into the nucleus; a process that was also efficiently inhibited by the treatment with GSH-C4. Overall, the evidence suggests that GSH decrement is required for efficient activation of an inflammatory condition and, at the same time, GSH-C4 can be envisaged as a good candidate to abrogate such process, expanding the anti-inflammatory role of this molecule in chronic inflammatory states.