COVID-19, corticosteroids and public health: a reappraisal.

OBJECTIVES: To assess whether regulatory guidance on the use of dexamethasone in hospitalised COVID-19 patients is applicable to the larger population of COVID-19 cases. The surge in worldwide demand for dexamethasone suggests that the guidance, although correct, has not emphasised the danger of its wider use. STUDY DESIGN: Data from the Randomised Evaluation of COVID-19 Therapy (RECOVERY) trial and the World Health Organisation (WHO) prospective meta-analysis have been deconstructed and analysed. METHODS: To provide context, relevant publications were identified in PubMed using the following keywords: COVID-19, RECOVERY trial, WHO meta-analysis, variants, immunity, public health. RESULTS: The WHO guidance 'Corticosteroids for COVID-19' was based on their prospective meta-analysis. This meta-analysis was weighted by data from the RECOVERY trial. CONCLUSIONS: In terms of COVID-19, dexamethasone has value in a narrow indication, namely, in hospitalised patients requiring respiratory support. The media blitz likely resulted in the wider use of dexamethasone in outpatients and as a preventive medication. This is reflected in the surge in worldwide demand for dexamethasone. We ask whether the use of steroids, beyond regulatory indications, may be responsible for the recent increase in mortality and especially the emergence of mucormycosis? From the public health standpoint, the current guidance for use of dexamethasone in COVID-19 could benefit from clarification and the addition of a cautionary note.

Pediatric Inflammatory Multisystem Syndrome (PIMS) - Potential role for cytokines such Is IL-6.

COVID-19 is a transmissible respiratory disease caused by coronavirus SARS-CoV-2, which is similar to SARS or MERS. Its increased severity was noted in aged patients usually over 65 years of age. Children and young people have an asymptomatic or mild course of the disease.Unfortunately, the number of children with problems after mild or asymptomatic COVID-19 recovery is increasing and their troubles resemble Kawasaki disease, although the laboratory findings seem to be different. This condition is called pediatric inflammatory multisystem syndrome (PIMS), and it is a new disease seen in children directly influenced by previous SARS-CoV-2 infection. The literature reports that PIMS typically follows 2-4 weeks after SARS-CoV-2 infection. The clinical symptoms of the affected children are extremely complex, ranging from gastrointestinal to cardiovascular problems with frequent skin and mucosal manifestations, and without intensive treatment they can be fatal. The exact causes of PIMS are recently unknown, however, it is explained as hyperactivation of immunity.In this minireview, we summarize data on the prominent role of the IL-6-IL-6R-STAT3 axis in PIMS aetiopathogenesis. Therapeutic manipulation of IL-6 or IL-6 receptor could be an approach to the treatment of children with severe PIMS.

Translation in solid cancer: are size-based response criteria an anachronism?

The purpose of translation is the development of effective medicinal products based on validated science. A parallel objective is to obtain marketing authorization for the translated product. Unfortunately, in solid cancer, these two objectives are not mutually consistent as evidenced by the contrast between major advances in science and the continuing dismal record of pharmaceutical productivity. If the problem is unrelated to science, then the process of translation may require a closer examination, namely, the criteria for regulatory approval. This realization is important because, in this context, the objective of translation is regulatory approval, and science does not passively translate into useful medicinal products. Today, in solid cancer, response criteria related to tumor size are less useful than during the earlier cytotoxic drugs era; advanced imaging and biomarkers now allow for tracking of the natural history of the disease in the laboratory and the clinic. Also, it is difficult to infer clinical benefit from tumor shrinkage since it is rarely sustained. Accordingly, size-based response criteria may represent an anachronism relative to translation in solid cancer and it may be appropriate to align preclinical and clinical effort and shift the focus to local invasion and metastasis. The shift from a cancer cellcentric model to a stroma centric model offers novel opportunities not only to interupt the natural history of the disease, but also to rethink the relevance of outdated criteria of clinical response. Current evidence favors the opinion that, in solid cancer, a different, broader, and contextual approach may lead to interventions that could delay local invasion and metastasis. All elements supporting this shift, especially advanced imaging, are in place(AU)

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Translation in solid cancer: are size-based response criteria an anachronism?

The purpose of translation is the development of effective medicinal products based on validated science. A parallel objective is to obtain marketing authorization for the translated product. Unfortunately, in solid cancer, these two objectives are not mutually consistent as evidenced by the contrast between major advances in science and the continuing dismal record of pharmaceutical productivity. If the problem is unrelated to science, then the process of translation may require a closer examination, namely, the criteria for regulatory approval. This realization is important because, in this context, the objective of translation is regulatory approval, and science does not passively translate into useful medicinal products. Today, in solid cancer, response criteria related to tumor size are less useful than during the earlier cytotoxic drugs era; advanced imaging and biomarkers now allow for tracking of the natural history of the disease in the laboratory and the clinic. Also, it is difficult to infer clinical benefit from tumor shrinkage since it is rarely sustained. Accordingly, size-based response criteria may represent an anachronism relative to translation in solid cancer and it may be appropriate to align preclinical and clinical effort and shift the focus to local invasion and metastasis. The shift from a cancer cell-centric model to a stroma centric model offers novel opportunities not only to interupt the natural history of the disease, but also to rethink the relevance of outdated criteria of clinical response. Current evidence favors the opinion that, in solid cancer, a different, broader, and contextual approach may lead to interventions that could delay local invasion and metastasis. All elements supporting this shift, especially advanced imaging, are in place.

Dynamics and morphology of focal adhesions in complex 3D environment.

Focal adhesions are specific types of cellular adhesion structures through which both mechanical force and regulatory signals are transmitted. Recently, the existence of focal adhesions in 3D environment has been questioned. Using a unique life-like model of dermis-based matrix we analysed the presence of focal adhesions in a complex 3D environment. Although the dermis-based matrix constitutes a 3D environment, the interface of cell-to-matrix contacts on thick bundled fibres within this matrix resembles 2D conditions. We call this a quasi-2D situation. We suggest that the quasi-2D interface of cell-to-matrix contacts constituted in the dermis-based matrix is much closer to in tissue conditions than the meshed structure of mostly uniform thin fibres in the gel-based matrices. In agreement with our assumption, we found that the cell adhesion structures are formed by cells that invade the dermis-based matrix and that these structures are of similar size as focal adhesions formed on fibronectin-coated coverslips (2D). In both 2D situation and the dermis-based matrix, we observed comparable vinculin dynamics in focal adhesions and comparable enlargement of the focal adhesions in response to a MEK inhibitor. We conclude that focal adhesions that are formed in the 3D environment are similar in size and dynamics as those seen in the 2D setting.

Fibronectin-replating experiment: procedure and analysis.

In this review protocols are described for studying protein tyrosine kinase signalling upon integrin-mediated cell adhesion. We have outlined detailed procedures for fibronectin-replating experiment, biochemical examination of the phosphotyrosine content of cellular proteins by immunoblotting using phosphorylation-specific antibodies or immunoprecipitation and analysis with general phosphotyrosine antibodies. Despite great advances that were made toward optimizing the described procedures, all these methods still remain in many respects an art, given the plentiful of variables and the extent to which the optimum conditions vary from one experimental condition to the other. Examples of performed experiments using the described procedures thus also include notes regarding variability of approaches based on experimental conditions.

Expression of protein tyrosine kinase pp60v-src variants in Dictyostelium discoideum.

We achieved production of v-Src of the low-oncogenic PRC and its variant proviral structure H19 in Dictyostelium discoideum, an emerging host system suitable for synthesis of heterologous proteins. To accomplish their expression, the first six codons of the N-terminus of v-src had to be changed according to the D. discoideum codon preference. Alternatively, N-terminal fusions of 6xHis-tag or GFP were sufficient to overcome the incompatibility in codon usage. D. discoideum-expressed v-Src kinases of the expected molecular weight were recognized by Src-specific antibodies; GFP-PRC was distributed uniformly in the cytosol. In contrast to other lower eukaryotes, where the accumulation of v-Src leads to growth inhibition, D. discoideum cells silenced the kinase activity of PRC-derived v-Src and showed no developmental or growth defects.

The regulatory region of Prague C v-Src inhibits the activity of the Schmidt-Ruppin A v-Src kinase domain.

Existing variants of the oncogene v-src differ in their transforming potential as well as in the range of their hosts. We compared the protein kinase activities of two Prague C v-Src variants (PRC and H19), reported to be of low oncogenic potential (Plachý et al., 1995), with the highly oncogenic Schmidt-Ruppin A v-Src (SRA). We employed in vitro kinase assays of affinity-purified proteins expressed in rabbit reticulocyte lysate and in S. cerevisiae. In both systems used, the specific kinase activity of the Prague C v-Src kinases amounted to only ca 20% of the activity of SRA. This positions the PRC Src close to activated c-Src, despite the lack of the regulatory C-terminal tail in PRC. We constructed chimeras between PRC and SRA v-Src and tested them for specific kinase activity in S. cerevisiae. Remarkably, the regulatory N-terminal part of PRC, when fused to the SRA-derived kinase domain, lowered the chimeras' PK activity to ca 20%, suggesting that it is the regulatory part of PRC that is responsible for its low phosphotransferase activity.

The fission yeast ortholog of the coregulator SKIP interacts with the small subunit of U2AF.

The mode of action of transcriptional coregulators may involve the recruitment of spliceosome components. Using the two-hybrid screen, we examined the interaction partners of spSNW1, the S. pombe ortholog of the human coregulator SNW1/SKIP/NCoA-62, and found it to interact with the small subunit of the splicing factor U2AF (spU2AF23). The interaction involves the C-terminal parts of spU2AF23 and spSNW1. Tagged variants of both proteins were expressed in S. pombe and the interaction was proved by coprecipitation in nuclear extracts. This interaction would explain the finding of SKIP in nuclear speckles (Mintz, P. J., et al., EMBO J. 18, 4308-4320, 1999) and in reconstituted spliceosomes (Neubauer, G., et al., Nat. Genet. 20, 46-50, 1998). We deleted the spSNW1 gene in the diploid strain and demonstrated that spSNW1 is an essential gene in S. pombe.