Double-blinded, randomised, placebo-controlled trial of convalescent plasma for COVID-19: analyses by neutralising antibodies homologous to recipients' variants.

INTRODUCTION: Convalescent plasma (CP) emerged as potential treatment for COVID-19 early in the pandemic. While efficacy in hospitalised patients has been lacklustre, CP may be beneficial at the first stages of disease. Despite multiple new variants emerging, no trials have involved analyses on variant-specific antibody titres of CP. METHODS: We recruited hospitalised COVID-19 patients within 10 days of symptom onset and, employing a double-blinded approach, randomised them to receive 200 ml convalescent plasma with high (HCP) or low (LCP) neutralising antibody (NAb) titre against the ancestral strain (Wuhan-like variant) or placebo in 1:1:1 ratio. Primary endpoints comprised intubation, corticosteroids for symptom aggravation, and safety assessed as serious adverse events. For a preplanned ad hoc analysis, the patients were regrouped by infused CP's NAb titers to variants infecting the recipients i.e. by titres of homologous HCP (hHCP) or LCP (hLCP). RESULTS: Of the 57 patients, 18 received HCP, 19 LCP and 20 placebo, all groups smaller than planned. No significant differences were found for primary endpoints. In ad hoc analysis, hHCPrecipients needed significantly less respiratory support, and appeared to be given corticosteroids less frequently (1/14; 7.1%) than those receiving hLCP (9/23; 39.1%) or placebo (8/20; 40%), (p = 0.077). DISCUSSION: Our double-blinded, placebo-controlled CP therapy trial remained underpowered and does not allow any firm conclusions for early-stage hospitalised COVID-19 patients. Interestingly, however, regrouping by homologous - recipients' variant-specific - CP titres suggested benefits for hHCP. We encourage similar re-analysis of ongoing/previous larger CP studies. TRIAL REGISTRATION: ClinTrials.gov identifier: NCT0473040.

Identification of the Nef-associated kinase as p21-activated kinase 2.

The Nef protein of primate immunodeficiency viruses plays an important role in the pathogenesis of acquired immunodeficiency syndrome (AIDS) [1] [2]. The interaction of Nef with the Nef-associated kinase (NAK) is one of the most conserved properties of different human and simian immunodeficiency virus (HIV and SIV) Nef alleles. The role of NAK association is currently not known but it has been implicated in enhanced viral infectivity in cell culture and in disease progression in SIV-infected macaques [3]. Previous studies have indicated that NAK shares many features with the p21-activated kinases (PAKs) [3], but the molecular identity of NAK has remained unknown. We have generated specific antisera against PAKs 1-3, and expressed these kinases individually as epitope-tagged proteins. By using these reagents in experiments involving partial proteolytic mapping, and exploiting the unique ability of PAK2 to serve as a caspase substrate, we have positively identified NAK as PAK2. Interestingly, although ectopic PAK2 overexpression efficiently replaced endogenous PAK2 from the complex with Nef, the total Nef-associated PAK2 activity was not increased, indicating the abundance of another cellular factor(s) as the limiting factor in Nef-PAK2 complex formation. Identification of NAK as PAK2 should now facilitate elucidation of its role as a mediator of the pathogenic effects of Nef.

A natural variability in the proline-rich motif of Nef modulates HIV-1 replication in primary T cells.

In the infected host, the Nef protein of HIV/SIV is required for high viral loads and thus disease progression. Recent evidence indicates that Nef enhances replication in the T cell compartment after the virus is transmitted from dendritic cells (DC). The underlying mechanism, however, is not clear. Here, we report that a natural variability in the proline-rich motif (R71T) profoundly modulated Nef-stimulated viral replication in primary T cells of immature dendritic cell/T cell cocultures. Whereas both Nef variants (R/T-Nef) downregulated CD4, only the isoform supporting viral replication (R-Nef) efficiently interacted with signaling molecules of the T cell receptor (TCR) environment and stimulated cellular activation. Structural analysis suggested that the R to T conversion induces conformational changes, altering the flexibility of the loop containing the PxxP motif and hence its ability to bind cellular partners. Our report suggests that functionally and conformationally distinct Nef isoforms modulate HIV replication on the interaction level with the TCR-signaling environment once the virus enters the T cell compartment.

Negative regulation of immunoglobulin kappa light-chain gene transcription by a short sequence homologous to the murine B1 repetitive element.

B-cell-specific expression of the immunoglobulin kappa light-chain (Ig kappa) gene is in part accomplished by negative regulatory influences. Here we describe a new negatively acting element (termed kappa NE) immediately upstream of the NF-kappa B-binding site in the Ig kappa intronic enhancer. The 27-bp kappa NE sequence is conserved in the corresponding positions in the rabbit and human Ig kappa genes, and the human kappa NE homolog was shown to have a similar negative regulatory activity. Data base searches using the mouse kappa NE sequence revealed a striking homology to murine B1 repetitive sequences. A sequence homologous to kappa NE and B1 was also noted in a previously identified silencer element in the murine T-cell receptor alpha locus. The homologous T-cell receptor alpha locus sequence, but notably not a corresponding 27-bp B1 consensus sequence, showed a negative regulatory potential similar to that of kappa NE. The negative effect of kappa NE by itself was not cell type specific but became so when paired with its 5'-flanking sequence in the Ig kappa enhancer. A short (30-bp) fragment upstream of kappa NE (termed kappa BS) was found to be necessary and sufficient for abolishing the negative effect of kappa NE in B cells. Point mutations in a T-rich motif within the kappa BS sequence allowed the transcriptional repression by kappa NE to be evident in B cells as well as other cells. As suggested by this cell-independent negative activity, proteins binding to the mouse and human kappa NE sequences were identified in all cell types tested.

Two N-myc polypeptides with distinct amino termini encoded by the second and third exons of the gene.

The N-myc and c-myc genes encode closely related nuclear phosphoproteins. We found that the N-myc protein from human tumor cell lines appears as four closely migrating polypeptide bands (p58 to p64) in sodium dodecyl sulfate-polyacrylamide gels. This and the recent finding that the c-myc protein is synthesized from two translational initiation sites located in the first and second exons of the gene (S. R. Hann, M. W. King, D. L. Bentley, C. W. Anderson, and R. N. Eisenman, Cell 52:185-195, 1988) prompted us to study the molecular basis of the N-myc protein heterogeneity. Dephosphorylation by alkaline phosphatase reduced the four polypeptide bands to a doublet with an electrophoretic mobility corresponding to the two faster-migrating N-myc polypeptides (p58 and p60). When expressed transiently in COS cells, an N-myc deletion construct lacking the first exon produced polypeptides similar to the wild-type N-myc protein, indicating that the first exon of the N-myc gene is noncoding. Furthermore, mutants deleted of up to two thirds of C-terminal coding domains still retained the capacity to produce a doublet of polypeptides, suggesting distinct amino termini for the two N-myc polypeptides. The amino-terminal primary structure of the N-myc protein was studied by site-specific point mutagenesis of the 5' end of the long open reading frame and by N-terminal radiosequencing of the two polypeptides. Our results show that the N-myc polypeptides are initiated from two alternative in-phase AUG codons located 24 base pairs apart at the 5' end of the second exon. Both of these polypeptides are phosphorylated and localized to the nucleus even when expressed separately. Interestingly, DNA rearrangements activating the c-myc gene are often found in the 1.7-kilobase-pair region between the two c-myc translational initiation sites and correlate with the loss of the longer c-myc polypeptide. Thus the close spacing of the two N-myc initiation codons could explain the relative resistance of the N-myc gene to similar modes of oncogenic activation.

Amplification of the c-myc oncogene in a subpopulation of human small cell lung cancer.

We have examined a panel of human lung cancer cell lines for amplification and expression of the c-myc, N-myc, and c-myb oncogenes. The cell lines analyzed represent various histopathological types of lung cancer: small cell carcinoma with neuroendocrine properties; squamous cell carcinoma with epithelial markers; and large cell carcinoma with a mixed neuroendocrine-epithelial phenotype. Two of six cell lines, both of which were small cell carcinomas, showed about a 20-fold amplification of the c-myc oncogene. In both cell lines, the amplification is accompanied by an enhanced expression of c-myc. The N-myc or c-myb genes were not amplified in any of the cell lines, nor were they expressed in detectable amounts. The results confirm and extend earlier findings on c-myc amplification in small cell lung cancer.

Expression of the L-myc gene is under positive control by short-lived proteins.

The c-myc oncogene is the most extensively studied member of the myc gene family which now consists of three characterized members, namely the c-myc, N-myc and L-myc genes. These genes are often found amplified in cell lines from small cell carcinomas of the lung (SCLC). In this study the L-myc gene was examined in a panel of human SCLC cell lines and tumors. One of the cell lines (U-1690) expressed abundant L-myc RNA and had an about 40-fold amplification of the L-myc gene. In contrast, no amplifications were found in clinical tumor material of ten SCLC specimens. The regulation of L-myc gene expression was studied with regard to mRNA stability and transcriptional control. The two L-myc transcripts observed in U-1690 cells were found to have different half-lives. Unlike c-myc mRNA in other cells, no significant stabilization of the L-myc mRNA occurred when protein synthesis was inhibited. Instead, transcription of the L-myc gene was found to be dependent on the synthesis of short-lived proteins.

Activation of protein kinase C increases phosphorylation of the L-myc trans-activator domain at a GSK-3 target site.

The L-myc protein migrates as three distinct differentially phosphorylated bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This phosphorylation can be rapidly increased either by treatment with the protein kinase C (PKC) activator phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or by inhibition of serine/threonine protein phosphatases with okadaic acid. In vitro mutagenesis and phosphoamino acid analyses define the N-terminal serine residues 38 and 42 of L-myc as critical targets for the PKC-dependent phosphorylation. These are the exclusive sites of phosphorylation in the N-terminal third of the L-myc protein, and can be phosphorylated in vitro by glycogen synthase kinase 3 beta (GSK-3 beta). A mutant L-myc protein in which these serines have been replaced by alanine residues does not show heterogeneous electrophoretic migration or hyperphosphorylation in response to PKC activation, and is not a substrate for GSK-3 beta in vitro. Similar potential phosphorylation sites are present in c-myc and N-myc in a highly conserved region thought to represent a transcriptional activation domain. We suggest that N-terminal phosphorylation of the L-myc protein is a means of rapid regulation of this oncoprotein, possibly mediated in vivo by the action of GSK-3.

SH3 domains with high affinity and engineered ligand specificities targeted to HIV-1 Nef.

The avid binding of HIV-1 Nef to the Src homology-3 (SH3) domain of Hck (KD 250 nM) has been shown to involve an interaction between the RT-loop of Hck-SH3 and residues in Nef outside of its prototypic polyproline type II (PPII) helix-containing SH3-ligand region. Such distinctive interactions are thought to provide specificity and affinity for other SH3/ligand protein complexes as well. Here, we have constructed and successfully displayed on the surface of M13 bacteriophage particles a complex library of SH3 domains, which are derived from Hck but carry a random hexapeptide substitution in their RT-loops (termed RRT-SH3). Using this strategy we have identified individual RRT-SH3 domains that can bind to Nef up to 40-fold more avidly than Hck-SH3. Some of these high-affinity RRT-SH3 domains resembled Hck-SH3 in that they bound much less well to a Nef variant containing an engineered F90R mutation that interferes with docking of the native Hck RT-loop. In addition, we could also select RRT-SH3 domains with an opposite specificity, which were dependent on the Arg90 residue for strong binding, and bound 100-fold less well to unmodified Nef. These results demonstrate the utility of phage-display in engineering of signaling protein interaction domains, and emphasize the importance of the RT-loop in SH3 ligand selection, thus suggesting a general strategy for creating SH3 domains with desired binding properties.

Induction of activator protein 1 (AP-1) in macrophages by human immunodeficiency virus type-1 NEF is a cell-type-specific response that requires both hck and MAPK signaling events.

Human immunodeficiency virus type 1 (HIV-1) Nef is important for viral infectivity and pathogenicity. HIV-1 infection is associated with inappropriate activation and defects in the function of monocytes/macrophages. We have studied the effects of HIV-1 Nef in the murine (RAW264.7) and human (THP-1) monocyte-macrophage cell lines. Investigation of the activator protein-1 (AP-1) transcription factor showed that Nef expression induced both its DNA binding and transcriptional activities. Increased AP-1 DNA binding activity in RAW264.7 cells was associated with raised levels of c-Fos expression and induction of mRNA for the AP-1 responsive tissue inhibitor of metalloproteinases-1 (TIMP-1) gene. Mutagenesis and kinase inhibition studies were employed to determine signaling pathways used by Nef to induce AP-1. Data from these studies indicated that induction of AP-1 by Nef is likely to be mediated through the MAPK (ERK1 and 2) signaling pathway and requires the proline-rich PxxP motif of Nef, suggesting the involvement of upstream protein kinases belonging to the Src family. Effects of Nef on AP-1 induction were cell lineage-specific, being stimulatory in macrophages, inhibitory in T cells and without effect in HeLa cells. These latter two observations led us to test the possibility that cell-specific interactions of Nef with Src family proteins may modulate AP-1 activity. To this end we demonstrated that a dominant-negative Hck mutant caused inhibition of Nef-mediated AP-1 DNA binding activity in RAW cells. In conclusion, induction of AP-1 by Nef is a specific feature of human and murine macrophage cell lines that requires signal transduction events involving Hck and MAPKs.

HIV-1 Nef and host cell protein kinases.

Nef is a 27-34 kD myristoylated protein unique to primate lentiviruses. A functional Nef gene is important for development of high viremia and simian AIDS in SIV infected rhesus macaques. Notably, animals infected with Nef-deleted attenuated viruses are resistant to subsequent challenge with pathogenic wild-type viruses. A critical role for Nef in development of AIDS in humans has been suggested by the observation that some individuals with a long-term nonprogressive HIV-1 infection (persons who show no clinical or immunological signs of immunodeficiency despite being HIV seropositive for over a decade) are infected with viruses carrying naturally occurring Nef deletions. The mechanism of Nef action remains incompletely understood, but multiple lines of evidence point out to a role in modulation of cellular signaling pathways via physical and functional interactions with host cell protein kinases. These findings will be discussed in the following, preceded by a short introduction into the role of Nef in cell biology of HIV infection, which is intended to serve as a critical review of our current understanding on this enigmatic issue rather than a comprehensive review of the literature.

Interactions of HIV-1 NEF with cellular signal transducing proteins.

Nef is a 27 - 34 kD myristoylated protein unique to primate lentiviruses. A functional Nef gene is important for development of high viremia and simian AIDS in SIV infected rhesus macaques (1). In a transgenic mouse model expression of Nef protein alone when expressed under a CD4-promoter is sufficient to cause an AIDS like disease (2). A critical role for Nef in development of AIDS in humans is suggested by the observation that some individuals with a long-term nonprogressive HIV-1 infection are infected with viruses carrying naturally occurring Nef deletions (3-5). The mechanism of Nef action remains incompletely understood, but multiple lines of evidence point out to a role in modulation of cellular signaling pathways via physical and functional interactions with host cell proteins.

Amplification and rearrangement of L-myc in human small-cell lung cancer.

DNA amplification of cellular proto-oncogenes is a well-established and common mechanism of oncogene activation in several types of human tumors, including the rapidly fatal small-cell lung cancer (SCLC). Approximately one fourth of primary SCLC tumors contain amplified copies of one of the three myc proto-oncogenes. Occasionally DNA amplification of the myc genes is associated with DNA rearrangements. Specifically, a novel locus named rlf is often involved in intrachromosomal L-myc rearrangements in SCLC. The structurally similar rearrangements are probably due to a highly repetitive region upstream of the L-myc gene, and result in the formation of a chimeric rlf-L-myc fusion protein. The consistent finding of the rlf-L-myc rearrangement in SCLC suggests that it may provide a selective advantage to the cells harboring it.

Macrophage metalloelastase (MME) as adjuvant for intra-tumoral injection of oncolytic adenovirus and its influence on metastases development.

Oncolytic adenoviruses are a promising treatment alternative for many advanced cancers, including colorectal cancer. However, clinical trials have demonstrated that single-agent therapy in advanced tumor masses is rarely curative. Poor spreading of the virus through tumor tissue is one of the major issues limiting efficacy. As oncolytic viruses kill preferentially cancer cells, high extracellular matrix (ECM) content constitutes potential barriers for viral penetration within tumors. In this study, the ECM-degrading proteases relaxin, hyaluronidase, elastase and macrophage metalloelastase (MME) were tested for their antitumor efficacy alone and in combination with oncolytic adenovirus. MME improved the overall antitumor efficacy of oncolytic adenovirus in subcutaneous HCT116 xenografts. In a liver metastatic colorectal cancer model, intra-tumoral treatment of primary tumors from HT29 cells with MME monotherapy or with oncolytic adenovirus inhibited tumor growth. Combination therapy showed no increased mortality in comparison with either monotherapy alone. Contradictory results of effects of MME on tumorigenesis and metastasis formation have been reported in the literature. This study demonstrates for the first time in a metastatic animal model that MME, as a monotherapy or in combination with oncolytic virus, does not increase tumor invasiveness. Co-administration of MME and oncolytic adenovirus may be a suitable approach for further optimization aiming at clinical applications for metastatic colorectal cancer.

Amplification of the N-myc oncogene in an adenocarcinoma of the lung.

c-myc oncogene is the most extensively studied member of the myc gene family, which now consists of three characterized members, namely the c-myc, N-myc, and L-myc genes. Deregulation owing to amplification and/or rearrangements of the c-myc gene have been described in a variety of human malignancies. Several neuroblastomas have amplifications of the N-myc genes. The c-myc, N-myc, or L-myc oncogenes are also found amplified in different cell lines from small cell carcinomas of the lung. In this study, we have examined the c-myc, N-myc, and c-erbB oncogenes in 34 clinical and autopsy tumor specimens representing various histopathological types of human lung cancer, including nine small cell lung cancers. A 30-fold amplification of the N-myc gene was found in a tumor histopathologically and histochemically verified as a typical adenocarcinoma. No amplifications of the c-myc or c-erbB oncogenes were seen in any of the tumors. In the DNA of one small cell carcinoma, an extra c-myc and N-myc cross-hybridizing restriction fragment was observed, possibly owing to an amplification of a yet uncharacterized myc-related gene.

Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4.

Human immunodeficiency virus (HIV) and simian immunodeficiency virus Nef proteins contain a conserved motif with the minimal consensus (PxxP) site for Src homology region 3 (SH3)-mediated protein-protein interactions. Nef PxxP motifs show specific binding to biotinylated SH3 domains of Hck and Lyn, but not to those of other tested Src family kinases or less related proteins. A unique cooperative role of a distant proline is also observed. Endogenous Hck of monocytic U937 cells can be specifically precipitated by matrix-bound HIV-1 Nef, but not by mutant protein lacking PxxP. Intact Nef PxxP motifs are dispensable for Nef-induced CD4 down-regulation, but are required for the higher in vitro replicative potential of Nef+ viruses. Thus, CD4 down-regulation and promotion of viral growth are two distinct functions of Nef, and the latter is mediated via SH3 binding.