Plitidepsin as a successful rescue treatment for prolonged viral SARS-CoV-2 replication in a patient with previous anti-CD20 monoclonal antibody-mediated B cell depletion and chronic lymphocytic leukemia.

BACKGROUND: There is an urgent need for highly efficacious antiviral therapies in immunosuppressed hosts who develop coronavirus disease (COVID-19), with special concern for those affected by hematological malignancies. CASE PRESENTATION: Here, we report the case of a 75-year-old male with chronic lymphocytic leukemia who was deficient in CD19+CD20+ B-lymphocyte populations due to previous treatment with anti-CD20 monoclonal antibodies. The patient presented with severe COVID-19 pneumonia due to prolonged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and was treated with two courses of the antiviral plitidepsin on a compassionate use basis. The patient subsequently achieved an undetectable viral load, and his pneumonia resolved. CONCLUSIONS: Treatment with plitidepsin was well-tolerated without any further hematological or cardiovascular toxicities. This case further supports plitidepsin as a potential antiviral drug in SARS-CoV-2 patients affected by immune deficiencies and hematological malignancies.

The marine sphingolipid-derived compound ES 285 triggers an atypical cell death pathway.

The isolation of new molecules from marine sources opens the door to their possible therapeutic use against tumors and other pathological conditions. Indeed, we recently defined the cytotoxicity of ES 285, obtained from the clam Mactromeris polynima, and its affects on the cells microfilament but not the microtubule network. Considering the analogy between ES 285 and sphingosine-related lipids, we wondered whether ES 285 might affect the activity of PKC at the intracellular level. While we anticipated that ES 285 might inhibit PKC, it turns out that in contrast it serves to activate PKC at the cellular level. Indeed, like other sphingosine-related lipids, ES 285 induces the phosphorylation of MARCKS. Additionally, we further examined the cytotoxicity of ES 285 to elucidate the molecular mechanisms through which this compound triggers apoptosis. When the influence of ES 285 on "cell death markers" was assessed, it became clear that ES285 activates caspase 3 and 12, and that it modified the phosphorylation of p53. In contrast, ES 285 does not affect other pathways widely implicated in regulating cell survival/apoptosis, such as JNK, Erks or Akt. Thus, these data suggest that ES 285-triggers an atypical cell death program when compared to other sphingosine-dependent apoptosis pathways.

The marine compound spisulosine, an inhibitor of cell proliferation, promotes the disassembly of actin stress fibers.

Spisulosine is a novel antiproliferative (antitumoral) compound of marine origin. In this work the molecular target for this toxic agent has been analyzed. In the presence of spisulosine, cultured cells change their morphology, first acquiring a fusiform morphology, and later becoming rounded without focal adhesions. Analysis of the cytoskeleton of treated cells indicate the absence of actin stress fibers.

Screening for new compounds with antiherpes activity.

A number of compounds have been tested for antiherpes activity. Actinobolin, amicetin, carrageenan, laspartomycin, megalomycin C, pleuromutilin, suramin and tetracenomycin C showed significant protection of HeLa cell monolayers infected with herpes simplex virus type 1. The action of these new antiherpes compounds was compared with those antiherpes agents that have been described previously. Actinobolin, amicetin and tetracenomycin C were also active against viruses other than herpes simplex.

Antibiotics that specifically block translation in virus-infected cells.

Several antibiotics including anthelmycin, blasticidin S, destomycin A, gougerotin, hygromycin B and edeine complex, known to powerfully block translation in cell-free systems, did neither inhibit protein synthesis in intact mouse L and 3T6 cells, nor in hamster BHK 21 cells, due to failure to cross the cell plasma membrane. However, after viral infection, these antibiotics exhibited a marked blockade of translation, that is related to the permeability changes induced by viral infection. The inhibition of protein synthesis by hygromycin B in virus-infected cells was studied over the time course of infection, both in encephalomyocarditis virus-infected mouse L cells and in Semliki forest virus-infected hamster BHK cells. We have observed that the entry of hygromycin B into virus-infected cells parallels the inhibition of cellular protein synthesis, i.e., the cells became permeable to this antibiotic at the time the shut-off of host translation occurred. A marked inhibition of picornavirus RNA synthesis by hygromycin B was also noticed, likely as a consequence of the inhibition of the viral replicase synthesis. Finally, a reduction in the virus yield by treatment of virus-infected cells with several antibiotics is also described. All these observations are considered in the context of the interference of viral infection with cellular functions and the potential use of inhibitors non-permeable to normal cells as antiviral agents.

A simple, one-step chromatographic procedure for the purification of lysozyme.

A rapid method for the purification of lysozyme (mucopeptide N-acetyl-muramoylhydrolase, EC 3.2.1.17) from hen egg-white has been devised. It was that gel filtration chromatography on agarose columns can be used selectively to purify lysozyme, due to the fact that this protein interacts with the agarose matrix and elutes later than the corresponding total volume for the column. Thus, lysozyme is directly obtained in a relatively pure form and with a high specific activity. In principle, this simple method can be used to prepare lysozymes from other sources.

L-glycerol-3-phosphate dehydrogenase from the insect Ceratitis capitata. Purfication, physicochemical and enzymic properties.

Soluble L-glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate: NAD+ 2-oxidoreductase, EC 1.1.1.8) from the mediterranean fruit fly Ceratitis capitata has been purified 130-fold with an overall yield of about 40%. The final preparation had a specific activity of about 200 mumol NADH/min/mg protein. The enzyme preparation has been shown to be homogeneous throughout disc gel electrophoresis, dodecyl sulphate gel electrophoresis, isoelectric focusing and ultracentrifugation. The Km values for dihydroxyacetone phosphate, NADH, L-glycerol-3-phosphate and NAD+ were respectively 0.33, 0.018, 0.74 and 0.26 mM. L-glycerol-3-phosphate dehydrogenase from the insect had a maximal activity around pH 6.6 for the oxidation of NADH and pH 10.0 for the reduction of NAD+. It was stable from pH 6.0 to pH 9.0 at 20 degrees C for 1 h and remained active after incubating at 30 degrees C for 30 min at pH 6.6. The enzyme was completely inactivated by incubating at 60 degrees C for 5 min. Enzyme stability versus ionic strength as well as the dependence of the reaction velocity on temperature are also reported. The active enzyme was found to have a minimum molecular weight of approx. 63 000. Molecular weight determinations by sodium dodecyl sulphate gel electrophoresis gave subunit weights of 33 500. The isoelectric point of the protein was determined by electrofocusing and found to be 5.75 +/- 0.05. The extinction coefficient at 278 nm was calculated by dry weight measurements to be E1cm 1mg/ml = 0.42 +/- 0.1. Sedimentation velocity studies on ultracentrifuge indicated a dependence of the sedimentation coefficient on the enzyme concentration. The amino acid composition of the enzyme was determined. The protein has no free N-terminal residue and the digestion with carboxypeptidases gave the C-terminal sequence: -ala-gly-ser. All these data are discussed in relation to the properties of the enzyme from other sources.

Lysozyme from the insect Ceratitis capitata eggs.

1. Lysozyme from eggs of the Dipterous Ceratitis capitata (Wiedeman) has been purified by ion-exchange chromatography and gel filtration and its physicochemical properties have been investigated. This is the first insect lysozyme characterized so far and it exhibits some properties different to those described for other animal lysozymes. 2. Lysozyme from the insect eggs has a molecular weight of about 23200 and a sedimentation coefficient of 2.4 S. Molecular weight determination by sodium dedecylsulphate gel electrophoresis indicates that the molecule consists of a single polypeptide chain. 3. This lysozyme preparation shows notable stability at acidic pH values and lability at alkline pH values. It shows a single optimum pH at about 6.5.4. Chitinase/muramidase specific activity ratio is around 350 times higher for the insect lysozyme than for the hen egg-white enzyme. 5. The amino-acid composition shows the presence of one tryptophan residue per molecule of enzyme. This fact differentiates the lysozyme from insect eggs from other animal and plant lysozymes. From the amino acid composition, the absorption coefficient and the partial specific volume are calculated. 6. Glycine is the N-terminal residue.

Primary structure of cytochrome c from the insect Ceratitis capitata.

The complete amino acid sequence of cytochrome c from the Dipterous Ceratitis capitata (serie Acalypterae) has been determined by combining automatic and manual methods of sequence analysis. No overlaps between positions 79 and 80, 86 and 87, 91 and 92 as well as between 99 and 100 were obtained. The alignment of these peptides was done by homology with other sequences of cytochromes c from insects already described. Comparison with the sequences of cytochromes c of other Diptera studied so far shows three changes (positions 50, 60 and 61, according to vertebrate cytochrome c numeration) from the Acalypteran Drosophila melanogaster and five changes (positions 9, 36, 50, 60 and 61) from that of the Calypteran Haematobia irritans.