Lethal mutagenesis, or virus extinction produced by enhanced mutation rates, is under investigation as an antiviral strategy that aims at counteracting the adaptive capacity of viral quasispecies, ...and avoiding selection of antiviral-escape mutants. To explore lethal mutagenesis of hepatitis C virus (HCV), it is important to establish whether ribavirin, the purine nucleoside analogue used in anti-HCV therapy, acts as a mutagenic agent during virus replication in cell culture. Here we report the effect of ribavirin during serial passages of HCV in human hepatoma Huh-7.5 cells, regarding viral progeny production and complexity of mutant spectra. Ribavirin produced an increase of mutant spectrum complexity and of the transition types associated with ribavirin mutagenesis, resulting in HCV extinction. Ribavirin-mediated depletion of intracellular GTP was not the major contributory factor to mutagenesis since mycophenolic acid evoked a similar decrease in GTP without an increase in mutant spectrum complexity. The intracellular concentration of the other nucleoside-triphosphates was elevated as a result of ribavirin treatment. Mycophenolic acid extinguished HCV without an intervening mutagenic activity. Ribavirin-mediated, but not mycophenolic acid-mediated, extinction of HCV occurred via a decrease of specific infectivity, a feature typical of lethal mutagenesis. We discuss some possibilities to explain disparate results on ribavirin mutagenesis of HCV.
Neuroprotective strategies aimed to pharmacologically treat stroke, a prominent cause of death, disability, and dementia, have remained elusive. A promising approach is restriction of excitotoxic ...neuronal death in the infarct penumbra through enhancement of survival pathways initiated by brain‐derived neurotrophic factor (BDNF). However, boosting of neurotrophic signaling after ischemia is challenged by downregulation of BDNF high‐affinity receptor, full‐length tropomyosin‐related kinase B (TrkB‐FL), due to calpain‐degradation, and, secondarily, regulated intramembrane proteolysis. Here, we have designed a blood–brain barrier (BBB) permeable peptide containing TrkB‐FL sequences (TFL457) which prevents receptor disappearance from the neuronal surface, early induced after excitotoxicity. In this way, TFL457 interferes TrkB‐FL cleavage by both proteolytic systems and increases neuronal viability via a PLCγ‐dependent mechanism. By preserving downstream CREB and MEF2 promoter activities, TFL457 initiates a feedback mechanism favoring increased levels in excitotoxic neurons of critical prosurvival mRNAs and proteins. This neuroprotective peptide could be highly relevant for stroke therapy since, in a mouse ischemia model, it counteracts TrkB‐FL downregulation in the infarcted brain, efficiently decreases infarct size, and improves neurological outcome.
Synopsis
During excitotoxicity, the neuroprotective peptide MTFL457 stabilizes the BDNF receptor TrkB‐FL. In a severe model of permanent brain ischemia (acute stroke), MTFL457 reduces infarct size and neurological damage, offering great potential for neurological conditions associated to excitotoxicity.
Downregulation of BDNF receptor TrkB‐FL by proteolysis, induced in human stroke and ischemia models, challenges BDNF therapies.
A blood‐brain barrier permeable peptide containing TrkB‐FL sequences (TFL457) able to prevent receptor processing was designed, the peptide effect correlating with increased neuronal viability after excitotoxic damage.
Primarily, TFL457 restrains the decrease of cell‐surface receptor induced by excitotoxicity that precedes TrkB‐FL processing. The preserved receptor maintains PLCγ‐dependent signaling and triggers a feedback survival mechanism mediated by CREB and MEF2 activities.
This neuroprotective peptide could be highly relevant for stroke therapy since, in a mouse model of ischemia, TFL457 counteracts TrkB‐FL downregulation, efficiently decreases infarct size and improves neurological outcome.
Combination of TFL457 with strategies aimed to increase BDNF availability might further improve this peptide efficiency in stroke and other excitotoxicity‐associated disorders, such as neurodegenerative diseases.
During excitotoxicity, the neuroprotective peptide MTFL457 stabilizes the BDNF receptor TrkB‐FL. In a severe model of permanent brain ischemia (acute stroke), MTFL457 reduces infarct size and neurological damage, offering great potential for neurological conditions associated to excitotoxicity.
Neuroprotective strategies aimed to pharmacologically treat stroke, a prominent cause of death, disability, and dementia, have remained elusive. A promising approach is restriction of excitotoxic ...neuronal death in the infarct penumbra through enhancement of survival pathways initiated by brain-derived neurotrophic factor (BDNF). However, boosting of neurotrophic signaling after ischemia is challenged by downregulation of BDNF high-affinity receptor, full-length tropomyosin-related kinase B (TrkB-FL), due to calpain-degradation, and, secondarily, regulated intramembrane proteolysis. Here, we have designed a blood-brain barrier (BBB) permeable peptide containing TrkB-FL sequences (TFL
) which prevents receptor disappearance from the neuronal surface, early induced after excitotoxicity. In this way, TFL
interferes TrkB-FL cleavage by both proteolytic systems and increases neuronal viability via a PLCγ-dependent mechanism. By preserving downstream CREB and MEF2 promoter activities, TFL
initiates a feedback mechanism favoring increased levels in excitotoxic neurons of critical prosurvival mRNAs and proteins. This neuroprotective peptide could be highly relevant for stroke therapy since, in a mouse ischemia model, it counteracts TrkB-FL downregulation in the infarcted brain, efficiently decreases infarct size, and improves neurological outcome.
Frailty is a common condition among critically ill patients. Usually evaluated in a mixed population of medical, cardiac and surgical patients, we aimed to assess the impact of frailty on short- and ...long-term mortality exclusively in critically ill older medical patients.
We included 285 patients aged≥70 years admitted to ICU (2009–2017). Comorbidities, severity scores, treatment intensity and complications were recorded. Pre-hospital frailty, measured by Clinical Frailty Scale (CFS), was defined as a score ≥ 5 according to this scale.
Prevalence of frailty (CFS ≥ 5) of 18.6%. Frail patients were more likely to be female (64.2% vs. 35.6%, p < .001) or suffer from heart failure (17% vs. 6%,p = .021). Apache II score was higher in frail than in non-frail patients (27.4 ± 7.1 vs. 24.8 ± 8.6,p = .041). Age, comorbidities, treatment intensity, complications, and ICU and hospital length of stay were similar between frail and non-frail patients. Life-sustaining treatment limitation was more frequent in frail patients (47.2% vs. 20.7%,p < .001). Except for ICU mortality, frailty was an independent predictor of short- and long-term mortality after adjustment for sociodemographic, comorbidities, severity scores, treatment intensity and complications.
Frailty (CFS ≥ 5) was independently associated with short- and long-term mortality in older patients admitted to ICU exclusively due to a medical reason.
•Frailty condition was exclusively evaluated in critically ill older medical patients (≥ 70 years).•Frailty (CFS ≥ 5) was associated with short- and long- term mortality after controlling for several variables.•ICU mortality is not a suitable form of assessing the impact of frailty in older critically ill patients admitted to ICU.•Frailty assessment should be routinely included in thedecision-making process of older medical patients admitted to ICU.
Background:
Cystoid Macular Edema (CME) is the most important cause of blindness in non-infectious uveitis (NIU)
(1).
Corticosteroids and conventional and/or biological immunosuppressant may be ...required
(1-6).
High-dose intravenous methylprednisolone (IVMP) pulse therapy may induce a rapid improvement.
Objectives:
To evaluate the efficacy and safety of IVMP pulse therapy in CME of different immune mediated inflammatory diseases (IMID).
Methods:
Multicentre study of 66 patients with severe ocular inflammation who received IVMP. The underlying diseases were: Vogt Koyanagy Harada disease (VKHD)(n=24), Behçet’s disease (BD) (19) and idiopathic NIU (23). The main outcome variable was macular thickness and macular edema (Optical coherence tomography OCT >300 μm); that was assessed at 0 (basal), 2-5, 7, 15 and 30 days after IVMP.
The results are expressed as mean ±SD for normally distributed variables, or as median IQR when are not. Comparison of continuous variables was performed using the Wilcoxon test.
Results:
We studied 43♀/ 23♂ patients. The main features are shown in TABLE 1. IVMP dose ranged from 250 to 1000 mg/day administered for 3-5 consecutive days, the dose was established according to the presence or not of other systemic manifestations apart from uveitis. All of them had active intraocular inflammation at the moment of the study.
Table 1.
Main features of 66 patients with cystoid macular edema.
VKHD
(n=24
)
Idiophatic
(n=23
)
Behcet’s disease
(n=19
)
Overall
(n=66
)
Men/Women, n
5/19
9/14
9/10
66
Mean age (years
)
42 ± 11
47 ± 15
33 ± 10
-
Unilateral/Bilateral, n (%
)
2 (8.3) /22 (91.7
)
10 (43.5) /13 (56.5)
4 (21) /15 (79)
16/50
Inflammatory ocular patterns, n (%
)
Posterior uveitis
6 (25)
9 (39.1)
3 (15.8)
18
Panuveitis
18 (75)
14 (60.9)
16 (84.2)
48
Laboratory data, n (%
)
ANA
2 (8.34)
2 (8.7)
0 (0)
4
HLA B27
0 (0)
4 (17.4)
0 (0)
4
HLA B29
0 (0)
1 (4.3)
0 (0)
1
HLA B51
0 (0)
5 (21.7)
8 (42)
13
A rapid and maintained statistically improvement was observed in OCT values in all underlying diseases (FIGURE 1
).
No major side effects were observed.
Conclusion:
High-dose IVMP pulse therapy is useful and safe in the prompt control of CME, regardless of the underlying IMID.
References:
1Vegas-Revenga N, et al. Am J Ophthalmol. 2019; 200:85-94. doi: 10.1016/j.ajo.2018.12.019.
2Calvo-Río V, et al. Clin Exp Rheumatol. 2014;32(4 Suppl 84): S54-7. PMID: 25005576.
3Santos-Gómez M, et al. Clin Exp Rheumatol. 2016;34(6 Suppl 102): S34-S40. PMID:27054359.
4Atienza-Mateo B, et al. Rheumatology (Oxford) 2018;57(5):856-864. doi: 10.1093/rheumatology/kex480.
5Atienza-Mateo B, et al. Arthritis Rheumatol. 2019; 71(12):2081-2089. doi: 10.1002/art.41026.
6Martín-Varillas JL, et al. Ophthalmology. 2018;125(9):1444-1451. doi: 10.1016/j.ophtha.2018.02.020.
Figure 1.
Improvement of OCT in 66 patients with cystoid macular edema.
Disclosure of Interests:
None declared
To characterize and describe clinical experience with childhood-onset non-infectious uveitis.
A multicenter retrospective multidisciplinary national web-based registry of 507 patients from 21 ...hospitals was analyzed. Cases were grouped as immune disease-associated (IMDu), idiopathic (IDIu) or ophthalmologically distinct. Characteristics of juvenile idiopathic arthritis-associated (non-HLA-B27-related) uveitis (JIAu), IDIu, and pars planitis (PP) were compared.
IMDu (62.3%) and JIAu (51.9%) predominated in young females; and IDIu (22.7%) and PP (13.6%) in older children, without sex imbalance. Ocular complications occurred in 45.3% of cases (posterior synechiae 28%, cataracts 16%, band keratopathy 14%, ocular hypertension 11% and cystoid macular edema 10%) and were associated with synthetic (86%) and biologic (65%) disease-modifying antirheumatic drug (DMARD) use. Subgroups were significantly associated (
< 0.05) with different characteristics. JIAu was typically anterior (98%), insidious (75%), in ANA-positive (69%), young females (82%) with fewer complications (31%), better visual outcomes, and later use of uveitis-effective biologics. In contrast, IDIu was characteristically anterior (87%) or panuveitic (12.1%), with acute onset (60%) and more complications at onset (59%: synechiae 31% and cataracts 9.6%) and less DMARD use, while PP is intermediate, and was mostly bilateral (72.5%), persistent (86.5%) and chronic (86.8%), with more complications (70%; mainly posterior segment and cataracts at last visit), impaired visual acuity at onset, and greater systemic (81.2%), subtenon (29.1%) and intravitreal (10.1%) steroid use.
Prognosis of childhood uveitis has improved in the "biologic era," particularly in JIAu. Early referral and DMARD therapy may reduce steroid use and improve outcomes, especially in PP and IDIu.
The renin-angiotensin system (RAS) has emerged as one of the essential links in the pathophysiology of vascular disease. Angiotensin (Ang) II, the main peptide of the RAS, was considered as a ...vasoactive hormone, but in the past years, this view has been modified to a growth factor that regulates cell proliferation/apoptosis and fibrosis. Recently, this view has been enlarged with a novel conceptAng II participates in the inflammatory response, acting as a proinflammatory mediator. In resident vascular cells, Ang II produces chemokines, cytokines, and adhesion molecules, which contribute to the migration of inflammatory cells into the tissue injury. Ang II is also a chemotactic and mitogenic factor for mononuclear cells. The molecular mechanisms of Ang II–induced vascular damage are mediated by the activation of transcription factors, redox signaling systems, and production of endogenous growth factors. In addition, other components of the RAS could also be involved in the pathogenesis of cardiovascular diseases. The Ang II degradation product Ang III shares some of its properties with Ang II, including chemotaxis and production of growth factors and chemokines. All these data clearly demonstrate that Ang II is a true cytokine, show the complexity of the RAS in pathological processes, and provide some mechanistic responses of the beneficial effects of the treatment with RAS blockers in cardiovascular diseases.
PURPOSESpectral-domain optical coherence tomography (SD-OCT) is the most useful tool to measure choroidal thickness (CT). CT may be increased in ocular and systemic diseases. However, there are ...concerns relating reproducibility and external validity of OCT. The aim of this study was to assess the inter-observer and intra-observer variability of manual OCT measurements. METHODSCT was manually measured in the central choroid of 40 eyes from 21 subjects (11 healthy and 10 with ankylosing spondylitis) using RTVue-100 OCT (Optovue Inc., Fremont, CA, EE.UU.). Measurements were performed by 9 independent ophthalmologists from 6 different centers. To assess the inter-observer variability, the intra-class correlation coefficient (ICC) method was calculated. Also, intra-observer variability was assessed in 2 of the ophthalmologists. RESULTSThe mean subfoveal CT was 364.9±85.1μm (range, 170 to 572). The inter-observer ICC was 0.823 (CI 95%, 0.749 to 0.888, p<0.001). The intra-observer ICCs were 0.885 (CI 95%, 0.783 to 0.939, p<0.001) and 0.925 (CI 95%, 0.859 to 0.960. p<0.001). CONCLUSIONSIn this study, manual measurements of CT with OCT showed a good concordance. These results suggest that manual OCT is a valid tool for multicenter studies.
Postsynaptic density protein-95 (PSD-95) is a multidomain protein critical to the assembly of signaling complexes at excitatory synapses, required for neuronal survival and function. However, ...calpain-processing challenges PSD-95 function after overactivation of excitatory glutamate receptors (excitotoxicity) in stroke, a leading cause of death, disability and dementia in need of efficient pharmacological treatments. A promising strategy is neuroprotection of the infarct penumbra, a potentially recoverable area, by promotion of survival signaling. Interference of PSD-95 processing induced by excitotoxicity might thus be a therapeutic target for stroke and other excitotoxicity-associated pathologies. Methods: The nature and stability of PSD-95 calpain-fragments was analyzed using in vitro assays or excitotoxic conditions induced in rat primary neuronal cultures or a mouse model of stroke. We then sequenced PSD-95 cleavage-sites and rationally designed three cell-penetrating peptides (CPPs) containing these sequences. The peptides effects on PSD-95 stability and neuronal viability were investigated in the cultured neurons, subjected to acute or chronic excitotoxicity. We also analyzed the effect of one of these peptides in the mouse model of stroke by measuring infarct size and evaluating motor coordination and balance. Results: Calpain cleaves three interdomain linker regions in PSD-95 and produces stable fragments corresponding to previously described PSD-95 supramodules (PDZ1-2 and P-S-G) as well as a truncated form SH3-GK. Peptide TP95414, containing the cleavage site in the PDZ3-SH3 linker, is able to interfere PSD-95 downregulation and reduces neuronal death by excitotoxicity. Additionally, TP95414 is delivered to mice cortex and, in a severe model of permanent ischemia, significantly improves the neurological outcome after brain damage. Conclusions: Interference of excitotoxicity-induced PSD-95-processing with specific CPPs constitutes a novel and promising therapeutic approach for stroke treatment.
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