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BACKGROUND: The systemic inflammatory syndrome called "cytokine storm" has been described in COVID-19 pathogenesis, contributing to disease severity. The analysis of cytokine and chemokine levels in the blood of 21 SARS-CoV-2 positive patients throughout the phases of the pandemic has been studied to understand immune response dysregulation and identify potential disease biomarkers for new treatments. The present work reports the cytokine and chemokine levels in sera from a small cohort of individuals primarily infected with SARS-CoV-2 during the first wave of the COVID-19 pandemic in Milan (Italy). RESULTS: Among the 27 cytokines and chemokines investigated, a significant higher expression of Interleukin-9 (IL-9), IP-10 (CXCL10), MCP-1 (CCL2) and RANTES (CCL-5) in infected patients compared to uninfected subjects was observed. When the change in cytokine/chemokine levels was monitored over time, from the hospitalization day to discharge, only IL-6 and IP-10 showed a significant decrease. Consistent with these findings, a significant negative correlation was observed between IP-10 and anti-Spike IgG antibodies in infected individuals. In contrast, IL-17 was positively correlated with the production of IgG against SARS-CoV-2. CONCLUSIONS: The cytokine storm and the modulation of cytokine levels by SARS-CoV-2 infection are hallmarks of COVID-19. The current global immunity profile largely stems from widespread vaccination campaigns and previous infection exposures. Consequently, the immunological features and dynamic cytokine profiles of non-vaccinated and primarily-infected subjects reported here provide novel insights into the inflammatory immune landscape in the context of SARS-CoV-2 infection, and offer valuable knowledge for addressing future viral infections and the development of novel treatments.
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COVID-19 , Quimiocinas , Citocinas , SARS-CoV-2 , Humanos , COVID-19/inmunología , COVID-19/sangre , COVID-19/epidemiología , Italia/epidemiología , SARS-CoV-2/inmunología , Femenino , Masculino , Persona de Mediana Edad , Citocinas/sangre , Anciano , Quimiocinas/sangre , Adulto , Síndrome de Liberación de Citoquinas/sangre , Síndrome de Liberación de Citoquinas/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Biomarcadores/sangre , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , PandemiasRESUMEN
The pandemic of coronavirus disease 19 (COVID-19), caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2), had severe repercussions for breast cancer patients. Increasing evidence indicates that SARS-CoV-2 infection may directly impact breast cancer biology, but the effects of SARS-CoV-2 on breast tumor cells are still unknown. Here, we analyzed the molecular events occurring in the MCF7, MDA-MB-231 and HCC1937 breast cancer cell lines, representative of the luminal A, basal B/claudin-low and basal A subtypes, respectively, upon SARS-CoV-2 infection. Viral replication was monitored over time, and gene expression profiling was conducted. We found that MCF7 cells were the most permissive to viral replication. Treatment of MCF7 cells with Tamoxifen reduced the SARS-CoV-2 replication rate, suggesting an involvement of the estrogen receptor in sustaining virus replication in malignant cells. Interestingly, a metagene signature based on genes upregulated by SARS-CoV-2 infection in all three cell lines distinguished a subgroup of premenopausal luminal A breast cancer patients with a poor prognosis. As SARS-CoV-2 still spreads among the population, it is essential to understand the impact of SARS-CoV-2 infection on breast cancer, particularly in premenopausal patients diagnosed with the luminal A subtype, and to assess the long-term impact of COVID-19 on breast cancer outcomes.
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Neoplasias de la Mama , COVID-19 , SARS-CoV-2 , Tamoxifeno , Replicación Viral , Humanos , Neoplasias de la Mama/virología , Neoplasias de la Mama/patología , COVID-19/virología , Femenino , SARS-CoV-2/fisiología , Línea Celular Tumoral , Tamoxifeno/farmacología , Células MCF-7 , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión GénicaRESUMEN
BACKGROUND: The viral main protease (Mpro) of SARS-CoV-2 has been recently proposed as a key target to inhibit virus replication in the host. Therefore, molecules that can bind the catalytic site of Mpro could be considered as potential drug candidates in the treatment of SARS-CoV-2 infections. Here we proposed the application of a state-of-the-art analytical platform which combines metabolomics and protein structure analysis to fish-out potential active compounds deriving from a natural matrix, i.e., a blueberry extract. METHODS: The experiments focus on finding MS covalent inhibitors of Mpro that contain in their structure a catechol/pyrogallol moiety capable of binding to the nucleophilic amino acids of the enzyme's catalytic site. RESULTS: Among the potential candidates identified, the delphinidin-3-glucoside showed the most promising results. Its antiviral activity has been confirmed in vitro on Vero E6 cells infected with SARS-CoV-2, showing a dose-dependent inhibitory effect almost comparable to the known Mpro inhibitor baicalin. The interaction of delphinidin-3-glucoside with the Mpro pocket observed was also evaluated by computational studies. CONCLUSIONS: The HRMS analytical platform described proved to be effective in identifying compounds that covalently bind Mpro and are active in the inhibition of SARS-CoV-2 replication, such as delphinidin-3-glucoside.
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Antocianinas , Antivirales , Arándanos Azules (Planta) , Proteasas 3C de Coronavirus , Extractos Vegetales , Inhibidores de Proteasas , SARS-CoV-2 , Arándanos Azules (Planta)/química , Antocianinas/farmacología , Antocianinas/química , Antivirales/farmacología , Antivirales/química , Chlorocebus aethiops , Células Vero , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Animales , Extractos Vegetales/farmacología , Extractos Vegetales/química , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , Tratamiento Farmacológico de COVID-19 , Humanos , Simulación del Acoplamiento Molecular , COVID-19/virología , GlucósidosRESUMEN
We evaluated the association between biomarkers and COVID-19 mortality. Baseline characteristics of 403 COVID-19 patients included sex and age; biomarkers, measured throughout the follow-up, included lymphocytes, neutrophils, ferritin, C-reactive protein, glucose, and LDH. Hazard ratios (HRs) and corresponding 95% credible intervals (CIs) were estimated through joint models (JMs) using a Bayesian approach. We fitted univariable (a single biomarker) and multivariable (all biomarkers) JMs. In univariable analyses, all biomarkers were significantly associated with COVID-19 mortality. In multivariable analysis, HRs were 1.78 (95% CI: 1.13-2.87) with a doubling of neutrophils levels, 1.49 (95% CI: 1.19-1.95) with a doubling of C-reactive protein levels, 2.66 (95% CI: 1.45-4.95) for an increase of 100 mg/dL of glucose, and 1.31 (95% CI: 1.12-1.55) for an increase of 100 U/L of LDH. No evidence of association was observed for lymphocytes and ferritin in multivariable analysis. Men had a higher COVID-19 mortality risk than women (HR = 1.75; 95% CI: 1.07-2.80) and age showed the strongest effect with a rapid increase from 60 years. These findings using JM confirm the usefulness of biomarkers in assessing COVID-19 severity and mortality. Monitoring trend patterns of such biomarkers can provide additional help in tailoring the appropriate care pathway.
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INTRODUCTION: Immunosuppression after kidney transplantation (KTx) exposes recipients to Human Polyomaviruses (HPyVs) infections, whose natural history is still misunderstood. METHODS: Allograft biopsies, and urine from 58 donor-recipient pairs were collected before KTx (T0) and 1 (T1), 15 (T2), 30 (T3), 60 (T4), 90 (T5), 180 (T6), 270 (T7), 360 (T8), and 540 (T9) days after transplant. Specimens were tested for JC (JCPyV) and BK (BKPyV), by quantitative Real-Time PCR. The course of post-KTx HPyVs viruria, and the association between JCPyV viruria in recipients and donors, were evaluated. RESULTS: HPyVs were detected in 3/58 (5.2%) allograft biopsies. HPyVs viruria was present in 29/58 (50%) donors and 41/58 (70.7%) recipients. JCPyV DNA was detected in 26/58 (44.8%) donors and 25/58 recipients (43.1%), 19 of whom received kidney from JCPyV positive donor, whereas BKPyV genome was detected in 3 (5.2%) donors and 22 (37.9%) recipients. The median time of JCPyV, and BKPyV first episode of replication was 1, and 171 days post KTx, respectively. At T0, JCPyV viruria of donors was associated with increased risk of JCPyV replication post-KTx; recipients with JCPyV positive donors showed lower risk of BKPyV replication post-KTx. CONCLUSIONS: The results suggested that JCPyV may be transmitted by allograft, and that its replication post KTx might prevent BKPyV reactivation. Future investigation regarding correlation between chronic exposure to immunosuppressive agents and HPyVs urinary replication are warranted.
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Trasplante de Riñón , Poliomavirus , Humanos , Poliomavirus/genética , Trasplante de Riñón/efectos adversos , Estudios Longitudinales , Riñón , Receptores de TrasplantesRESUMEN
BACKGROUND: Human-cytomegalovirus (hCMV) infection involving the gastrointestinal tract represents a leading cause of morbidity and mortality among kidney transplant (KT) recipients (KTRs). Signs and symptoms of the disease are extremely variable. Prompt anti-viral therapy administration and immunosuppression modification are key factors for optimizing management. However, complex work-up strategies are generally required to confirm the preliminary diagnosis. Unfortunately, solid evidence and guidelines on this specific topic are not available. We consequently aimed to summarize current knowledge on post-KT hCMV-related gastrointestinal disease (hCMV-GID). METHODS: We conducted a systematic review (PROSPERO ID: CRD42023399363) about hCMV-GID in KTRs. RESULTS: Our systematic review includes 52 case-reports and ten case-series, published between 1985 and 2022, collectively reporting 311 cases. The most frequently reported signs and symptoms of hCMV-GID were abdominal pain, diarrhea, epigastric pain, vomiting, fever, and GI bleeding. Esophagogastroduodenoscopy and colonoscopy were the primary diagnostic techniques. In most cases, the preliminary diagnosis was confirmed by histology. Information on anti-viral prophylaxis were extremely limited as much as data on induction or maintenance immunosuppression. Treatment included ganciclovir and/or valganciclovir administration. Immunosuppression modification mainly consisted of mycophenolate mofetil or calcineurin inhibitor minimization and withdrawal. In total, 21 deaths were recorded. Renal allograft-related outcomes were described for 26 patients only. Specifically, reported events were acute kidney injury (n = 17), transplant failure (n = 5), allograft rejection (n = 4), and irreversible allograft dysfunction (n = 3). CONCLUSIONS: The development of local and national registries is strongly recommended to improve our understanding of hCMV-GID. Future clinical guidelines should consider the implementation of dedicated diagnostic and treatment strategies.
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Infecciones por Citomegalovirus , Enfermedades Gastrointestinales , Trasplante de Riñón , Humanos , Trasplante de Riñón/efectos adversos , Trasplante de Riñón/métodos , Citomegalovirus , Antivirales/uso terapéutico , Infecciones por Citomegalovirus/diagnóstico , Infecciones por Citomegalovirus/tratamiento farmacológico , Infecciones por Citomegalovirus/etiología , Ganciclovir/uso terapéutico , Enfermedades Gastrointestinales/diagnóstico , Enfermedades Gastrointestinales/tratamiento farmacológico , Enfermedades Gastrointestinales/etiologíaRESUMEN
(1) Background. Exploring the evolution of SARS-CoV-2 load and clearance from the upper respiratory tract samples is important to improving COVID-19 control. Data were collected retrospectively from a laboratory dataset on SARS-CoV-2 load quantified in leftover nasal pharyngeal swabs (NPSs) collected from symptomatic/asymptomatic individuals who tested positive to SARS-CoV-2 RNA detection in the framework of testing activities for diagnostic/screening purpose during the 2020 and 2021 winter epidemic waves. (2) Methods. A Statistical approach (quantile regression and survival models for interval-censored data), novel for this kind of data, was applied. We included in the analysis SARS-CoV-2-positive adults >18 years old for whom at least two serial NPSs were collected. A total of 262 SARS-CoV-2-positive individuals and 784 NPSs were included: 193 (593 NPSs) during the 2020 winter wave (before COVID-19 vaccine introduction) and 69 (191 NPSs) during the 2021 winter wave (all COVID-19 vaccinated). We estimated the trend of the median value, as well as the 25th and 75th centiles of the viral load, from the index episode (i.e., first SARS-CoV-2-positive test) until the sixth week (2020 wave) and the third week (2021 wave). Interval censoring methods were used to evaluate the time to SARS-CoV-2 clearance (defined as Ct < 35). (3) Results. At the index episode, the median value of viral load in the 2021 winter wave was 6.25 log copies/mL (95% CI: 5.50-6.70), and the median value in the 2020 winter wave was 5.42 log copies/mL (95% CI: 4.95-5.90). In contrast, 14 days after the index episode, the median value of viral load was 3.40 log copies/mL (95% CI: 3.26-3.54) for individuals during the 2020 winter wave and 2.93 Log copies/mL (95% CI: 2.80-3.19) for those of the 2021 winter wave. A significant difference in viral load shapes was observed among age classes (p = 0.0302) and between symptomatic and asymptomatic participants (p = 0.0187) for the first wave only; the median viral load value is higher at the day of episode index for the youngest (18-39 years) as compared to the older (40-64 years and >64 years) individuals. In the 2021 epidemic, the estimated proportion of individuals who can be considered infectious (Ct < 35) was approximately half that of the 2020 wave. (4) Conclusions. In case of the emergence of new SARS-CoV-2 variants, the application of these statistical methods to the analysis of virological laboratory data may provide evidence with which to inform and promptly support public health decision-makers in the modification of COVID-19 control measures.
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COVID-19 , Adulto , Humanos , Adolescente , COVID-19/diagnóstico , COVID-19/epidemiología , SARS-CoV-2/genética , Vacunas contra la COVID-19 , ARN Viral , Estudios Retrospectivos , FaringeRESUMEN
Breastmilk protects newborns from infections through specific and nonspecific compounds. This study investigated the neutralizing activity against SARS-CoV-2 of breastmilk from SARS-CoV-2 negative, unvaccinated mothers, and compared it to that from infected nursing mothers. We enrolled women after COVID-19 swab testing results upon maternity admission, and divided them into two groups: group A, COVID-19-positive mothers, and group B, negative mothers. Breastmilk was randomly sampled at 2, 7, and 20 days postpartum. We collected 19 samples for Group A and 41 for Group B. A microneutralization assay was used to determine the 50% neutralization (NT50) titre. The presence of neutralizing antibodies was also determined. Group A had 100% neutralizing samples at 2 days postpartum (T0), declining 7 days postpartum (T1) and 20 days postpartum (T2). Group B samples exhibited neutralizing activity mostly at 7 days postpartum (T1) (90%). Negative mothers' samples showed no correlation between NT50 titres and antibodies' presence, suggesting that non-specific breastmilk components may exert antiviral action against SARS-CoV-2.
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COVID-19 , Leche Humana , Recién Nacido , Embarazo , Femenino , Humanos , SARS-CoV-2 , Lactancia , Madres , Anticuerpos NeutralizantesRESUMEN
The careful tailoring of the most appropriate immunosuppressive strategy for recipients of a kidney transplant (KT) regularly faces a risk of complications that may harm the actual graft and affect patient survival [...].
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Rapid detection of whole virus particles in biological or environmental samples represents an unmet need for the containment of infectious diseases. Here, an optical device enabling the enumeration of single virion particles binding on antibody or aptamers immobilized on a surface with anti-reflective coating is described. In this regime, nanoparticles adhering to the sensor surface provide localized contributions to the reflected field that become detectable because of their mixing with the interfering waves in the reflection direction. Thus, these settings are exploited to realize a scan-free, label-free, micro-array-type digital assay on a disposable cartridge, in which the virion counting takes place in wide field-of-view imaging. With this approach we could quantify, by enumeration, different variants of SARS-CoV-2 virions interacting with antibodies and aptamers immobilized on different spots. For all tested variants, the aptamers showed larger affinity but lower specificity relative to the antibodies. It is found that the combination of different probes on the same surface enables increasing specificity of detection and dynamic range.
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Aptámeros de Nucleótidos , Técnicas Biosensibles , COVID-19 , Humanos , SARS-CoV-2 , Técnicas Biosensibles/métodos , Anticuerpos , ViriónRESUMEN
Alongside vaccines, antiviral drugs are becoming an integral part of our response to the SARS-CoV-2 pandemic. Nirmatrelvir-an orally available inhibitor of the 3-chymotrypsin-like cysteine protease-has been shown to reduce the risk of progression to severe COVID-19. However, the impact of nirmatrelvir treatment on the development of SARS-CoV-2-specific adaptive immune responses is unknown. Here, by using mouse models of SARS-CoV-2 infection, we show that nirmatrelvir administration blunts the development of SARS-CoV-2-specific antibody and T cell responses. Accordingly, upon secondary challenge, nirmatrelvir-treated mice recruited significantly fewer memory T and B cells to the infected lungs and mediastinal lymph nodes, respectively. Together, the data highlight a potential negative impact of nirmatrelvir treatment with important implications for clinical management and might help explain the virological and/or symptomatic relapse after treatment completion reported in some individuals.
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Inmunidad Adaptativa , Antivirales , Tratamiento Farmacológico de COVID-19 , Lactamas , Animales , Ratones , COVID-19/inmunología , SARS-CoV-2 , Antivirales/administración & dosificación , Inmunidad Adaptativa/efectos de los fármacos , Lactamas/administración & dosificación , Células T de Memoria/inmunología , Linfocitos B/inmunología , Ratones Endogámicos C57BLRESUMEN
Lysozyme (E.C. 3.2.1.17), an about 14 kDa protein and pI 11, widely spread in nature, is present in humans mainly in milk, saliva, and intestinal mucus as a part of innate defense mechanisms. It is endowed with antimicrobial activity due to its action as an N-acetylmuramidase, cleaving the 1-4ß glycosidic linkage in the peptidoglycan layer of Gram-positive bacteria. This antimicrobial activity is exerted only against a limited number of Gram-negative bacteria. Different action mechanisms are proposed to explain its activity against Gram-negative bacteria, viruses, and fungi. The antiviral activity prompted the study of a possible application of lysozyme in the treatment of SARS-CoV-2 infections. Among the different sources of lysozyme, the chicken egg albumen was chosen, being the richest source of this protein (c-type lysozyme, 129 amino acids). Interestingly, the activity of lysozyme hydrochloride against SARS-CoV-2 was related to the heating (to about 100 °C) of this molecule. A chemical-physical characterization was required to investigate the possible modifications of native lysozyme hydrochloride by heat treatment. The FTIR analysis of the two preparations of lysozyme hydrochloride showed appreciable differences in the secondary structure of the two protein chains. HPLC and NMR analyses, as well as the enzymatic activity determination, did not show significant modifications.
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COVID-19 , Muramidasa , Humanos , Muramidasa/química , Calor , SARS-CoV-2/metabolismo , Bacterias Gramnegativas/metabolismo , Antivirales/farmacologíaRESUMEN
The ongoing emergency provoked by the SARS-CoV-2 pandemic demands the development of technologies to mitigate the spread of infection, and UV irradiation is a technique that can efficiently address this issue. However, proper use of UV equipment for disinfection requires an understanding of how the effects on SARS-CoV-2 are dependent on certain parameters. In this work, we determined the UV-C inactivation constant k for SARS-CoV-2 using an LED source at λ = 280 nm. Specifically, a Log3 reduction was measured after irradiation for 24 min with a delivered UV-C dose of 23 J m-2 . By multitarget model fitting, n = 2 and k = 0.32 ± 0.02 m2 J-1 were obtained. A lag time for the inactivation effect was also observed, which was attributed to the low irradiation levels used to perform the study. The combination of k and delay time allows for reliable estimation of disinfection times in small, closed environments.
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COVID-19 , SARS-CoV-2 , Humanos , COVID-19/prevención & control , Rayos Ultravioleta , Desinfección/métodos , Pandemias/prevención & control , Inactivación de Virus/efectos de la radiaciónRESUMEN
SARS-CoV-2 variants of concern (VOCs) represent an alarming threat as they show altered biological behavior and may escape vaccination effectiveness. Broad-spectrum antivirals could play an important role to control infections. The activity of Echinacea purpurea (Echinaforce® extract, EF) against (i) VOCs B1.1.7 (alpha), B.1.351.1 (beta), P.1 (gamma), B1.617.2 (delta), AV.1 (Scottish), B1.525 (eta), and B.1.1.529.BA1 (omicron); (ii) SARS-CoV-2 spike (S) protein-pseudotyped viral particles and reference strain OC43 as well as (iii) wild type SARS-CoV-2 (Hu-1) was analyzed. Molecular dynamics (MD) were applied to study the interaction of Echinacea's phytochemical markers with known pharmacological viral and host cell targets. EF extract broadly inhibited the propagation of all investigated SARS-CoV-2 VOCs as well as the entry of SARS-CoV-2 pseudoparticles at EC50's ranging from 3.62 to 12.03 µg/mL. The preventive addition of 25 µg/mL EF to epithelial cells significantly reduced sequential infection with SARS-CoV-2 (Hu-1) and OC43. MD analyses showed constant binding affinities to VOC-typical S protein variants for alkylamides, caftaric acid, and feruloyl-tartaric acid in EF extract and interactions with serine protease TMPRSS-2. EF extract demonstrated stable virucidal activity across seven tested VOCs, likely due to the constant affinity of the contained phytochemical substances to all spike variants. A possible interaction of EF with TMPRSS-2 partially would explain the cell protective benefits of the extract by the inhibition of membrane fusion and cell entry. EF may therefore offer a supportive addition to vaccination endeavors in the control of existing and future SARS-CoV-2 virus mutations.
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Since the declaration of the COVID-19 pandemic, the number of kidney transplants (KT) performed worldwide has plummeted. Besides the generalised healthcare crisis, this unprecedented drop has multiple explanations such as the risk of viral transmission through the allograft, the perceived increase in SARS-CoV-2-related morbidity and mortality in immunocompromised hosts, and the virtual "safety" of dialysis while awaiting effective antiviral prophylaxis or treatment. Our institution, operating at the epicentre of the COVID-19 pandemic in Italy, has continued the KT programme without pre-set limitations. In this single-centre retrospective observational study with one-year follow-up, we assessed the outcomes of patients who had undergone KT (KTR) or remained on the transplant waiting list (TWL), before (Pre-COV) or during (COV) the pandemic. The main demographic and clinical characteristics of the patients on the TWL or receiving a KT were very similar in the two periods. The pandemic did not affect post-transplant recipient and allograft loss rates. On the contrary, there was a trend toward higher mortality among COV-TWL patients compared to Pre-COV-TWL subjects. Such a discrepancy was primarily due to SARS-CoV-2 infections. Chronic exposure to immunosuppression, incidence of delayed allograft function, and rejection rates were comparable. However, after one year, COV-KTR showed significantly higher median serum creatinine than Pre-COV-KTR. Our data confirm that KT practice could be safely maintained during the COVID-19 pandemic, with excellent patient- and allograft-related outcomes. Strict infection control strategies, aggressive follow-up monitoring, and preservation of dedicated personnel and resources are key factors for the optimisation of the results in case of future pandemics.
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Current knowledge on Leishmania infection after kidney transplantation (KT) is limited. In order to offer a comprehensive guide for the management of post-transplant Leishmaniasis, we performed a systematic review following the latest PRISMA Checklist and using PubMed, Scopus, and Embase as databases. No time restrictions were applied, including all English-edited articles on Leishmaniasis in KT recipients. Selected items were assessed for methodological quality using a modified Newcastle-Ottawa Scale. Given the nature and quality of the studies (case reports and retrospective uncontrolled case series), data could not be meta-analyzed. A descriptive summary was therefore provided. Eventually, we selected 70 studies, describing a total of 159 cases of Leishmaniasis. Most of the patients were adult, male, and Caucasian. Furthermore, they were frequently living or travelling to endemic regions. The onset of the disease was variable, but more often in the late transplant course. The clinical features were basically similar to those reported in the general population. However, a generalized delay in diagnosis and treatment could be detected. Bone marrow aspiration was the preferred diagnostic modality. The main treatment options included pentavalent antimonial and liposomal amphotericin B, both showing mixed results. Overall, the outcomes appeared as concerning, with several patients dying or losing their transplant.
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More than two years have passed since the viral outbreak that led to the novel infectious respiratory disease COVID-19, caused by the SARS-CoV-2 coronavirus. Since then, the urgency for effective treatments resulted in unprecedented efforts to develop new vaccines and to accelerate the drug discovery pipeline, mainly through the repurposing of well-known compounds with broad antiviral effects. In particular, antiparasitic drugs historically used against human infections due to protozoa or helminth parasites have entered the main stage as a miracle cure in the fight against SARS-CoV-2. Despite having demonstrated promising anti-SARS-CoV-2 activities in vitro, conflicting results have made their translation into clinical practice more difficult than expected. Since many studies involving antiparasitic drugs are currently under investigation, the window of opportunity might be not closed yet. Here, we will review the (controversial) journey of these old antiparasitic drugs to combat the human infection caused by the novel coronavirus SARS-CoV-2.
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BACKGROUND: Human polyomavirus BK (BKPyV) is the etiologic agent of polyomavirus-associated nephropathy, a leading cause of kidney transplant dysfunction. Because of the lack of antiviral therapies, immunosuppression minimization is the recommended treatment. This strategy offers suboptimal outcomes and entails a significant risk of rejection. Our aim was to evaluate the effect of different immunosuppressive drugs (leflunomide, tacrolimus, mycophenolic acid, sirolimus, and everolimus) and their combinations in an in vitro model of BKPyV infection. METHODS: Human renal tubular epithelial cells were infected with BKPyV and treated with leflunomide, tacrolimus, mycophenolic acid, sirolimus, and everolimus, administered alone or in some combination thereof. Viral replication was assessed every 24 hours (up to 72 hours) by BKPyV-specific quantitative real-time polymerized chain reaction for the VIRAL PROTEIN 1 sequence in cell supernatants and by western blot analysis targeting the viral protein 1 and the glyceraldehyde 3-phosphate dehydrogenase on total protein lysates. Results were described as viral copies/mL and compared between treatments at any prespecified time point of the study. RESULTS: The highest inhibitory effects were observed using leflunomide or everolimus plus mycophenolic acid (mean BKPyV replication log reduction 0.28). The antiviral effect of everolimus persisted when it was used in combination with tacrolimus (mean BKPyV replication log reduction 0.27). CONCLUSIONS: Our experience confirms that everolimus has anti-BKPyV properties and prompts future research to investigate possible mechanisms of action. It also provides a rational basis for targeted clinical trials evaluating alternative immunosuppressive modification strategies.
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Virus BK , Trasplante de Riñón , Infecciones por Polyomavirus , Infecciones Tumorales por Virus , Humanos , Inmunosupresores/farmacología , Inmunosupresores/uso terapéutico , Tacrolimus/farmacología , Tacrolimus/uso terapéutico , Infecciones por Polyomavirus/tratamiento farmacológico , Leflunamida/farmacología , Leflunamida/uso terapéutico , Everolimus/farmacología , Everolimus/uso terapéutico , Ácido Micofenólico/farmacología , Ácido Micofenólico/uso terapéutico , Trasplante de Riñón/efectos adversos , Sirolimus/farmacología , Sirolimus/uso terapéutico , Antivirales/farmacología , Antivirales/uso terapéutico , Proteínas ViralesRESUMEN
In the novel pandemic of Coronavirus Disease 2019, high levels of pro-inflammatory cytokines lead to endothelial activation and dysfunction, promoting a pro-coagulative state, thrombotic events, and microvasculature injuries. The aim of the present work was to investigate the effect of SARS-CoV-2 on pro-inflammatory cytokines, tissue factor, and chemokine release, with Human Microvascular Endothelial Cells (HMEC-1). ACE2 receptor expression was evaluated by western blot analysis. SARS-CoV-2 infection was assessed by one-step RT-PCR until 7 days post-infection (p.i.), and by Transmission Electron Microscopy (TEM). IL-6, TNF-α, IL-8, IFN-α, and hTF mRNA expression levels were detected by RT-PCR, while cytokine release was evaluated by ELISA. HMEC-1 expressed ACE2 receptor and SARS-CoV-2 infection showed a constant viral load. TEM analysis showed virions localized in the cytoplasm. Expression of IL-6 at 24 h and IFN-α mRNA at 24 h and 48 h p.i. was higher in infected than uninfected HMEC-1 (p < 0.05). IL-6 levels were significantly higher in supernatants from infected HMEC-1 (p < 0.001) at 24 h, 48 h, and 72 h p.i., while IL-8 levels were significantly lower at 24 h p.i. (p < 0.001). These data indicate that in vitro microvascular endothelial cells are susceptible to SARS-CoV-2 infection but slightly contribute to viral amplification. However, SARS-CoV-2 infection might trigger the increase of pro-inflammatory mediators.
Asunto(s)
COVID-19 , Enzima Convertidora de Angiotensina 2 , Quimiocinas/genética , Quimiocinas/metabolismo , Citocinas/metabolismo , Células Endoteliales/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , SARS-CoV-2RESUMEN
INTRODUCTION: The ongoing coronavirus disease 19 (COVID-19) outbreak involves the pediatric population, but to date, few reports have investigated the circulation of variants among children. MATERIAL AND METHODS: In this retrospective study, non-hospitalized pediatric patients with SARS-CoV-2-positive nasopharyngeal swabs (NPS) were enrolled at the Institute for Maternal and Child Health-IRCCS Burlo Garofolo, Trieste (Italy), from November 2020 to January 2022. SARS-CoV-2 variants were identified by in vitro viral isolation, amplification, automatic sequencing of the receptor binding domain (RBD) of the SARS-CoV-2 spike coding gene, and subsequent next-generation sequencing. The growth curves of the isolated strains were defined in vitro by infecting Vero-E6 cells and quantifying the viral load in the supernatants up to 72 h post-infection by qRT-PCR. The neutralization activity of sera obtained from a COVID-19 vaccinated subject, recovered (2020) patient, vaccinated and recovered (2021) patient, and seronegative subject was assessed by microneutralization assay against the different variants. RESULTS: In total, 32 SARS-CoV-2-positive children, 16 (50%) females, with a median age of 1.4 years (range: 1 day-13 years), were enrolled. The D614G amino acid substitution was detected in all isolated and amplified viral strains. Of the 32 isolates, 4 (12.5%) carried a nonsynonymous nucleotide mutation leading to the N439K (3/4), lineage B.1.258 (∆H69/∆V70), and S477N (1/4) substitution. In 7/32 (21.8%) isolates, amino acid substitutions allowed the identification of a delta variant, lineage B.1.617.2-AY.43, and in 1/32 (3.1%), the Omicron strain (B.1.1.529.BA1) was identified. The growth curves of the B.1, B.1.258 (∆H69/∆V70), B.1.617.2-AY.43, and B.1.1.529.BA1 variants did not show any significant differences. A reduction in the serum neutralizing activity against B.1.258 (∆H69/∆V70) only in a vaccinated subject (1.7-fold difference), against B.1.617.2-AY.43 in a vaccinated subject and in recovered patients (12.7 and ≥2.5-fold differences, respectively), and against B.1.1.529.BA1 variant (57.6- and 1.4-fold differences in vaccinated and in vaccinated and recovered patients) were observed compared to the B.1 variant. CONCLUSIONS: SARS-CoV-2 variants carrying the B.1.258 (∆H69/∆V70) and S477N substitutions were reported here in a pediatric population for the first time. Although the growth rates of the isolated strains (B.1.258, B.1.617.2-AY.43, B.1.1.529.BA1) did not differ from the B.1 variant, neutralizing activity of the sera from vaccinated subjects significantly decreased against these variants. Attention should be devoted to the pediatric population to prevent the spread of new SARS-CoV-2 variants in an unvaccinated and predominantly naive population.