High levels of pro-inflammatory SARS-CoV-2-specific biomarkers revealed by in vitro whole blood cytokine release assay (CRA) in recovered and long-COVID-19 patients.

BACKGROUND: Cytokines induced by SARS-CoV-2 infection play a crucial role in the pathophysiology of COVID-19 and hyperinflammatory responses have been associated with poor clinical outcomes, with progression to severe conditions or long-term subacute complications named as long-COVID-19. METHODS: In this cross-sectional study, we aimed to evaluate a set of antigen-specific inflammatory cytokines in blood from recovered COVID-19 individuals or who suffered a post-acute phase of SARS-CoV-2 infection compared to healthy individuals with no history of COVID-19 exposition or infection. Interferon-gamma (IFN-γ), IFN-γ-induced protein 10 (IP-10), tumor necrosis factor (TNF), IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, and IL-17A were quantified by multiplex cytometric bead assay and enzyme-linked immunosorbent assay after stimulation of whole blood with recombinant Spike protein from SARS-CoV-2. Additionally, all participants have evaluated for anti-(S) protein-specific IgG antibodies. Clinical specimens were collected within two months of COVID-19 diagnosis. RESULTS: A total of 47 individuals were enrolled in the study, a median age of 43 years (IQR = 14.5), grouped into healthy individuals with no history of infection or exposure to SARS-CoV-2 (unexposed group; N = 21); and patients from the Health Complex of the Rio de Janeiro State University (UERJ), Brazil, who were SARS-CoV-2 positive by RT-PCR (COVID-19 group)-categorized as recovered COVID-19 (N = 11) or long-COVID-19 (N = 15). All COVID-19 patients presented at least one signal or symptom during the first two weeks of infection. Six patients were hospitalized and required invasive mechanical ventilation. Our results showed that COVID-19 patients had significantly higher levels of IFN-γ, TNF, IL-1ß, IL-2, IL-6, IL-8, and IP-10 than the unexposed group. The long-COVID-19 group has presented significantly high levels of IL-1ß and IL-6 compared to unexposed individuals, but not from recovered COVID-19. A principal-component analysis demonstrated 84.3% of the total variance of inflammatory-SARS-CoV-2 response in the first two components, and it was possible to stratify IL-6, TNF, IL-1ß, IL-10, and IL-2 as the top-five cytokines which are candidates to discriminate COVID-19 group (including long-COVID-19 subgroup) and healthy unexposed individuals. CONCLUSION: We revealed important S protein-specific differential biomarkers in individuals affected by COVID-19, bringing new insights into the inflammatory status or SARS-CoV-2 exposition determination.

Low doses of 3-phenyl-lawsone or meglumine antimoniate delivery by tattooing route are successful in reducing parasite load in cutaneous lesions of Leishmania (Viannia) braziliensis-infected hamsters.

Current therapeutic ways adopted for the treatment of leishmaniasis are toxic and expensive including parasite resistance is a growing problem. Given this scenario, it is urgent to explore treatment alternatives for leishmaniasis. The aim of this study was to evaluate the effect of 3-phenyl-lawsone (3-PL) naphthoquinone on Leishmania (Viannia) braziliensis infection, both in vitro and in vivo, using two local routes of administration: subcutaneous (higher dose) and tattoo (lower dose). In vitro 3-PL showed low toxicity for macrophages (CC50 >3200 µM/48h) and activity against intracellular amastigotes (IC50 = 193 ± 19 µM/48h) and promastigotes (IC50 = 116 ± 26 µM/72h), in which induced increased ROS generation. Additionally, 3-PL up-regulated the production of cytokines such as tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein 1 (MCP-1), interleukin-6 (IL-6) and IL-10 in infected macrophages. However, the anti-amastigote action was independent of nitric oxide production. Treatment of hamsters infected with L. (V.) braziliensis from one week after infection with 3-PL by subcutaneous (25 µg/Kg) or tattooing (2.5 µg/Kg) route, during 3 weeks (3 times/week) or 2 weeks (2 times/week) significantly decreased the parasite load (p<0.001) in the lesion. The reduction of parasite load by 3-PL treatment was comparable to reference drug meglumine antimoniate administered by the same routes (subcutaneous 1mg/Kg and tattoo 0.1mg/Kg). In addition, treatment started from five weeks after infection with 3-PL per tattoo also decreased the parasite load. These results show the anti-leishmanial effect of 3-PL against L. (V.) braziliensis and its efficacy by subcutaneous (higher dose) and tattoo (lower dose) routes. In addition, this study shows that drug delivery by tattooing the lesion allows the use of lower doses than the conventional subcutaneous route, which may support the development of a new therapeutic strategy that can be adopted for leishmaniasis.

Second-generation pterocarpanquinones: synthesis and antileishmanial activity

Despite the development of new therapies for leishmaniasis, among the 200 countries or territories reporting to the WHO, 87 were identified as endemic for Tegumentary Leishmaniasis and 75 as endemic for Visceral Leishmaniasis. The identification of antileishmanial drug candidates is essential to fill the drug discovery pipeline for leishmaniasis. In the hit molecule LQB-118 selected, the first generation of pterocarpanquinones was effective and safe against experimental visceral and cutaneous leishmaniasis via oral delivery. In this paper, we report the synthesis and antileishmanial activity of the second generation of pterocarpanoquinones. Methods: The second generation of pterocarpanquinones 2a-f was prepared through a palladium-catalyzed oxyarylation of dihydronaphtalen and chromens with iodolawsone, easily prepared by iodination of lawsone. The spectrum of antileishmanial activity was evaluated in promastigotes and intracellular amastigotes of L. amazonensis, L. braziliensis, and L. infantum. Toxicity was assessed in peritoneal macrophages and selective index calculated by CC50/IC50. Oxidative stress was measured by intracellular ROS levels and mitochondrial membrane potential in treated cells. Results: In this work, we answered two pertinent questions about the structure of the first-generation pterocarpanquinones: the configuration and positions of rings B (pyran) and C (furan) and the presence of oxygen in the B ring. When rings B and C are exchanged, we noted an improvement of the activity against promastigotes and amastigotes of L. amazonensis and promastigotes of L. infantum. As to the oxygen in ring B of the new generation, we observed that the oxygenated compound 2b is approximately twice as active against L. braziliensis promastigotes than its deoxy derivative 2a. Another modification that improved the activity was the addition of the methylenedioxy group. A variation in the susceptibility among species was evident in the clinically relevant form of the parasite, the intracellular amastigote. L. amazonensis was the species most susceptible to novel derivatives, whilst L. infantum was resistant to most of them. The pterocarpanoquinones (2b and 2c) that possess the oxygen atom in ring B showed induction of increased ROS production. Conclusions: The data presented indicate that the pterocarpanoquinones are promising compounds for the development of new leishmanicidal agents.(AU)

CK2 Secreted by Leishmania braziliensis Mediates Macrophage Association Invasion: A Comparative Study between Virulent and Avirulent Promastigotes.

CK2 is a protein kinase distributed in different compartments of Leishmania braziliensis: an externally oriented ecto-CK2, an intracellular CK2, and a secreted CK2. This latter form is constitutively secreted from the parasite (CsCK2), but such secretion may be highly enhanced by the association of specific molecules, including enzyme substrates, which lead to a higher enzymatic activity, called inductively secreted CK2 (IsCK2). Here, we examined the influence of secreted CK2 (sCK2) activity on the infectivity of a virulent L. braziliensis strain. The virulent strain presented 121-fold higher total CK2 activity than those found in an avirulent strain. The use of specific CK2 inhibitors (TBB, DRB, or heparin) inhibited virulent parasite growth, whereas no effect was observed in the avirulent parasites. When these inhibitors were added to the interaction assays between the virulent L. braziliensis strain and macrophages, association index was drastically inhibited. Polyamines enhanced sCK2 activity and increased the association index between parasites and macrophages. Finally, sCK2 and the supernatant of the virulent strain increased the association index between the avirulent strain and macrophages, which was inhibited by TBB. Thus, the kinase enzyme CK2 seems to be important to invasion mechanisms of L. braziliensis.