To evaluate the cumulative incidence, risk factors, and outcomes of COVID-19 in patients with Cushing's disease (CD).
In all, 60 patients with CD following up in our outpatient clinic answered via ...phone interview a questionnaire about the occurrence of COVID-19 infection documented by RT-PCR (including the diagnosis date and clinical outcome) and vaccination status. Clinical and biochemical data on disease activity (hypercortisolism) and comorbidities (obesity, diabetes mellitus, and hypertension) were obtained from the patients' electronic medical records. Risk ratios (RRs) of risk factors were obtained using univariate and multivariate analyses.
The cumulative incidence of COVID-19 in patients with CD during the observation period was 31.7%, which was higher than that in the general reference population (9.5%). The cumulative incidence of COVID-19 was significantly higher in patients with hypercortisolism (57% versus 17% in those without hypercortisolism, p = 0.012) and obesity (54% versus 9% in those without obesity, p < 0.001) but not in patients with hypertension or diabetes mellitus. On multivariate analysis, hypercortisolism and obesity were each independent risk factors for COVID-19 (RR 2.18, 95% CI 1.06-4.46, p = 0.033 and RR 5.19, 95% CI 1.61-16.74, p = 0.006, respectively).
The incidence of COVID-19 in patients with CD was associated with hypercortisolism, as expected, and obesity, a novel and unexpected finding. Thus, correction of hypercortisolism and obesity should be implemented in patients with CD during the current and future COVID-19 outbreaks.
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•HIV provirus in infected cells can be inactivated with CRISPR–Cas9.•Combinatorial CRISPR–Cas9 attack can prevent viral escape.•Both mutation and excision of HIV DNA contribute to ...virus inactivation.•HIV genetic diversity may compromise CRISPR–Cas9 efficiency.
Current antiretroviral drugs can efficiently block HIV replication and prevent transmission, but do not target the HIV provirus residing in cells that constitute the viral reservoir. Because drug therapy interruption will cause viral rebound from this reservoir, HIV-infected individuals face lifelong treatment. Therefore, novel therapeutic strategies are being investigated that aim to permanently inactivate the proviral DNA, which may lead to a cure. Multiple studies showed that CRISPR–Cas9 genome editing can be used to attack HIV DNA. Here, we will focus on not only how this endonuclease attack can trigger HIV provirus inactivation, but also how virus escape occurs and this can be prevented.
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely explored for sequence-specific attack on HIV-1 proviral DNA. We recently identified dual-guide RNA ...(dual-gRNA) combinations that can block HIV-1 replication permanently in infected cell cultures and prevent viral escape. Although the gRNAs were designed to target highly conserved viral sequences, their efficacy may be challenged by high genetic variation in the HIV-1 genome. We therefore evaluated the breadth of these dual-gRNA combinations against distinct HIV-1 isolates, including several subtypes. Replication of nearly all virus isolates could be prevented by at least one gRNA combination, which caused inactivation of the proviral genomes and the gradual loss of replication-competent virus over time. The dual-gRNA efficacy was not affected by most single nucleotide (nt) mismatches between gRNA and the viral target. However, 1-nt mismatches at the Cas9 cleavage site and two mismatches anywhere in the viral target sequence significantly reduced the inhibitory effect. Accordingly, sequence analysis of viruses upon breakthrough replication revealed the acquisition of escape mutations in perfectly matching and most 1-nt mismatching targets, but not in targets with a mismatch at the Cas9 cleavage site or with two mismatches. These results demonstrate that combinatorial CRISPR-Cas9 treatment can cure T cells infected by distinct HIV-1 isolates, but even minor sequence variation in conserved viral target sites can affect the efficacy of this strategy. Successful cure attempts against isolates with divergent target sequences may therefore require adaptation of the gRNAs.
Although several studies demonstrated that the HIV proviral DNA can be effectively targeted and inactivated by the CRISPR-Cas9 system, the precise inactivation mechanism has not yet been analyzed. ...Whereas some studies suggested efficient proviral DNA excision upon dual-gRNA/Cas9 treatment, we previously demonstrated that hypermutation of the target sites correlated with permanent virus inactivation. To better understand the mechanism underlying HIV inactivation, we analyzed the proviral DNA upon Cas9 attack with gRNA pairs. We observed that dual-gRNA targeting resulted more frequently in target site mutation than fragment excision, while fragment inversion was rarely observed. The frequencies varied for different gRNA combinations without an obvious relationship with the distance between the target sites, indicating that other gRNA and target DNA characteristics influence the DNA cleavage and repair processes.
Huntington's disease (HD) is a neurodegenerative disorder caused by a dominantly inherited CAG repeat expansion in the huntingtin gene (HTT). Neuroinflammation and microglia have been implicated in ...HD pathology, however it has been unclear if mutant HTT (mHTT) expression has an adverse cell-autonomous effect on microglial function, or if they are only activated in response to the neurodegenerative brain environment in HD. To establish a human cell model of HD microglia function, we generated isogenic controls for HD patient-derived induced pluripotent stem cells (iPSC) with 109 CAG repeats (Q109). Q109 and isogenic Q22 iPSC, as well as non-isogenic Q60 and Q33 iPSC lines, were differentiated to iPSC-microglia. Our study supports a model of basal microglia dysfunction in HD leading to elevated pro-inflammatory cytokine production together with impaired phagocytosis and endocytosis capacity, in the absence of immune stimulation. These findings are consistent with early microglia activation observed in pre-manifest patients and indicate that mHTT gene expression affects microglia function in a cell-autonomous way.
Chronic hepatitis B virus (HBV) infection is the result of an inadequate immune response towards the virus. Myeloid dendritic cells (mDC) of patients with chronic HBV are impaired in their maturation ...and function, resulting in more tolerogenic rather than immunogenic responses, which may contribute to viral persistence. The mechanism responsible for altered mDC function remains unclear. The HBV-infected patients display large amounts of HBV particles and viral proteins in their circulation, especially the surface antigen HBsAg, which allows multiple interactions between the virus, its viral proteins and DC. To assess whether HBV directly influences mDC function, the effects of HBV and HBsAg on human mDC maturation and function were investigated in vitro. As already described for internalization of HBV by DC, the present study shows that peripheral blood-derived mDC of healthy controls also actively take up HBsAg in a time-dependent manner. Cytokine-induced maturation in the presence of HBV or HBsAg resulted in a significantly more tolerogenic mDC phenotype as demonstrated by a diminished up-regulation of costimulatory molecules and a decreased T-cell stimulatory capacity, as assessed by T-cell proliferation and interferon-γ production. In addition, the presence of HBV significantly reduced interleukin-12 production by mDC. These results show that both HBV particles and purified HBsAg have an immune modulatory capacity and may directly contribute to the dysfunction of mDC in patients with chronic HBV. The direct immune regulatory effect of HBV and circulating HBsAg particles on the function of DC can be considered as part of the mechanism by which HBV escapes immunity.
To report a descriptive analysis of the virological results of Influenza-like illness (ILI) surveillance, comparing the frequency of detection of respiratory viruses during the pre-pandemic ...(2018-2019) and SARS-CoV-2 pandemic (2020-2021) winter seasons in Lombardy (Northern Italy).
From week 46 to week 17 of the following year, nasal-pharyngeal swabs (NPS) collected from ILIs were tested for influenza viruses (IV), RSV, rhinovirus and enterovirus with specific real-time RT-PCR assays. In 2020-2021, SARS-CoV-2 molecular detection was also included.
464 and 446 NPS were collected in 2018-2019 and 2020-2021, respectively. Sex distribution was similar between these two seasons (males: 51.5% vs 47%, P=0.165), while mean age was statistically higher in 2020-2021 (28.2 years IQR: 40.5 vs. 43.6 years IQR: 32.6, P<0.0001). ILI incidence was significantly higher in 2018/19 compared with 2020/21 season season (5.4/1,000 inhabitants vs. 1.9/1,000 inhabitants, P< 0.0001.ILI cases <15 years were less reported in 2020-2021 than in 2018-2019 season (P<0.0001). Contrary, those >15 years were more reported in 2020-2021 than in 2018-2019 (15-44 years, P=0.008; 45-64 years, P=0.002; ≥65 years, P<0.0001). In 2018-2019, IV and RSV accounted for 55% and 10% of ILIs, whereas in 2020-2021 no circulation of these viruses was observed. Enterovirus detections decreased from 3.4% in 2018-2019 to 0.4% in 2020-2021 (P=0.01), while rhinoviruses were detected at higher frequency in the latter season (8.6% vs. 11.9%, P=0.07). SARS-CoV-2 was detected in 25.3% of NPS in 2020-2021.
Since the beginning of the pandemic, non-pharmaceutical measures, including use of face-masks, was massively applied to contrast SARS-CoV-2 spread. This dramatically reduced the incidence of ILI, the circulation of SARS-CoV-2 and other respiratory viruses (such as IV and RSV) transmitted by droplets, but did not reduce rhinovirus detection, which continued to circulate in 2020-2021 season. ILIs surveillance has demonstrated to be able to capture changes in the epidemiology of respiratory viruses and it should be sustained and improved to increase the capture of ILI remained with unknown etiology.
This study evaluates whether intrathecal MVIIA injection after spinal cord injury (SCI) elicits neuroprotective effects. The test rats were randomly distributed into six groups- sham, placebo, MVIIA ...2.5 μM, MVIIA 5 μM, MVIIA 10 μM, and MVIIA 20 μM-and were administered the treatment four hours after SCI. After the optimal MVIIA dose (MVIIA 10 μM) was defined, the best time for application, one or four hours, was analyzed. Locomotor hind limb function and side effects were assessed. Forty-eight hours after the injury and immediately after euthanasia, spinal cord segments were removed from the test rats. Cell viability, reactive oxygen species, lipid peroxidation, and glutamate release were investigated. To examine the MVIIA mechanism of action, the gene expressions of pro-apoptotic (Bax, nNOS, and caspase-3, -8, -9, -12) and anti-apoptotic (Bcl-xl) factors in the spinal cord tissue samples were determined by real-time PCR, and the activities of antioxidant enzymes were also investigated. Application of intrathecal MVIIA 10 μM four hours after SCI prompted a neuroprotective effect: neuronal death decreased (22.46%), oxidative stress diminished, pro-apoptotic factors (Bax, nNOS, and caspase-3, -8) were expressed to a lesser extent, and mitochondrial viability as well as anti-apoptotic factor (Bcl-xl) expression increased. These results suggested that MVIIA provided neuroprotection through antioxidant effects. Indeed, superoxide dismutase (188.41%), and glutathione peroxidase (199.96%), reductase (193.86%), and transferase (175.93%) expressions increased. Therefore, intrathecal MVIIA (MVIIA 10 μM, 4 h) application has neuroprotective potential, and the possible mechanisms are related to antioxidant agent modulation and to intrinsic and extrinsic apoptotic pathways.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A previously healthy 6-year-old boy was admitted to hospital with hypotonia and hyposthenia of lower limbs. Electromyography and slow motor nerve conduction velocity test identified a lower limb ...acute motor axonal neuropathy. Brain and spinal cord magnetic resonance imaging demonstrated multifocal cortical gray matter lesions in both cerebral hemispheres consistent with gray matter acute disseminated encephalitis otherwise with viral/
Mycoplasma pneumoniae
encephalitis, and signs of involvement of anterior nerve roots of the cauda equina consistent with Guillain-Barré syndrome. The patient resulted negative to routinely bacterial and viral investigations but positive to human parechovirus that sequence analyses confirmed as type 6. Intravenous immunoglobulins and methylprednisolone treatment were administered but did not relieve the symptoms of Guillain-Barré syndrome. The disease improved gradually over the next 3-month follow-up with a complete remission of both central and peripheral nervous system symptoms.
The value of SARS-CoV-2 monitoring in urban wastewater samples (WWS) for surveillance of virus spread at a population-wide level has been largely demonstrated. Aim of this study was to optimize an ...analytical workflow to detect SARS-CoV-2 RNA in WWS and to monitor SARS-CoV-2 spread during the first wave of COVID-19 epidemic (March–June 2020) in Lombardy, northern Italy.
The workflow consisted in WWS concentration by using PEG-8000 precipitation, a modified RNA extraction (QIAamp MinElute Virus Spin Kit; QIAGEN) and a one-step real-time RT-PCR detecting two portions of the N gene of SARS-CoV-2. Composite 24-hour WWS were collected once a week at the inlet of 8 wastewater treatment plants (WWTPs) with an overall catchment of 2,276,000 inhabitants, located in representative COVID-19 hotspots in Lombardy, from the end of March to mid-June 2020. 107 WWS were obtained and analysed. SARS-CoV-2 RNA copies/L/WWS were multiplied by the flow rate of each WWTP (m3/day) and the obtained load (copies/day/1,000 people) was normalized to the number of inhabitants served by WWTPs.
The optimized workflow allowed to identify 1E+3 copies/mL of SARS-CoV-2 in concentrated WWS with a turnaround time of 8 hours. Overall, the presence of SARS-CoV-2 RNA was identified in 65/107 WWS (61%). The highest rate of positive WWS (78.7%; 26/33) was identified in the Bergamo province, that was the epicentre during the first wave of COVID-19 epidemic (March-June 2020) in Lombardy. The highest amount of SARS-CoV-2 RNA was identified in late March/early April, when the overall viral load reflecting the number of individuals shedding the virus ranged from 9.3E+10 copies/day/1,000 people to 8.2E+8 copies/day/1,000 people. Since the end of May, WWS tested negative to SARS-CoV-2 detection.
According to the epidemiological features of the first wave of SARS-CoV-2 epidemic in Lombardy, the highest amount of SARS-CoV-2 RNA was detected in WWS collected in the areas most affected by COVID-19 (i.e. Bergamo province). This optimized workflow of WWS surveillance can help assessing the real number of individuals – both symptomatic and asymptomatic – able to spread the virus and appraising the effect of preventive measures.
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