Levels of soluble complement regulators predict severity of COVID-19 symptoms.

The SARS-CoV-2 virus continues to cause significant morbidity and mortality worldwide from COVID-19. One of the major challenges of patient management is the broad range of symptoms observed. While the majority of individuals experience relatively mild disease, a significant minority of patients require hospitalisation, with COVID-19 still proving fatal for some. As such, there remains a desperate need to better understand what drives this severe disease, both in terms of the underlying biology, but also to potentially predict at diagnosis which patients are likely to require further interventions, thus enabling better outcomes for both patients and healthcare systems. Several lines of evidence have pointed to dysregulation of the complement cascade as a major factor in severe COVID-19 outcomes. How this is underpinned mechanistically is not known. Here, we have focussed on the role of the soluble complement regulators Complement Factor H (FH), its splice variant Factor H-like 1 (FHL-1) and five Factor H-Related proteins (FHR1-5). Using a targeted mass spectrometry approach, we quantified these proteins in a cohort of 188 plasma samples from controls and SARS-CoV-2 patients taken at diagnosis. This analysis revealed significant elevations in all FHR proteins, but not FH, in patients with more severe disease, particularly FHR2 and FHR5 (FHR2: 1.97-fold, p<0.0001; FHR5: 2.4-fold, p<0.0001). Furthermore, for a subset of 77 SARS-CoV-2 +ve patients we also analysed time course samples taken approximately 28 days post-diagnosis. Here, we see complement regulator levels drop in all individuals with asymptomatic or mild disease, but regulators remain high in those with more severe outcomes, with elevations in FHR2 over baseline levels in this group. These data support the hypothesis that elevation of circulating levels of the FHR family of proteins could predict disease severity in COVID-19 patients, and that the duration of elevation (or lack of immune activation resolution) may be partly responsible for driving poor outcomes in COVID-19.

Lymphocytes from chronic lymphocytic leukaemia undergo ABL1-linked amoeboid motility and homotypic interaction as an early adaptive change to ex vivo culture.

BACKGROUND: Those stimuli that together promote the survival, differentiation and proliferation of the abnormal B-lymphocytes of chronic lymphocytic leukaemia (CLL) are encountered within tissues, where together they form the growth-supporting microenvironment. Different tissue-culture systems promote the survival of the neoplastic lymphocytes from CLL, partly replicating the in vivo tissue environment of the disorder. In the present study, we focussed on the initial adaptive changes to the tissue culture environment focussing particularly on migratory behaviour and cellular interactions. METHODS: A high-density CLL culture system was employed to test CLL cell-responses using a range of microscopic techniques and flow cytometric analyses, supported by mathematical measures of cell shape-change and by biochemical techniques. The study focussed on the evaluation of changes to the F-actin cytoskeleton and cell behaviour and on ABL1 signalling processes. RESULTS: We showed that the earliest functional response by the neoplastic lymphocytes was a rapid shape-change caused through rearrangement of the F-actin cytoskeleton that resulted in amoeboid motility and promoted frequent homotypic interaction between cells. This initial response was functionally distinct from the elongated motility that was induced by chemokine stimulation, and which also characterised heterotypic interactions between CLL lymphocytes and accessory cells at later culture periods. ABL1 is highly expressed in CLL lymphocytes and supports their survival, it is also recognised however to have a major role in the control of the F-actin cytoskeleton. We found that the cytoplasmic fraction of ABL1 became co-localised with F-actin structures of the CLL lymphocytes and that the ABL1 substrate CRKL became phosphorylated during initial shape-change. The ABL-inhibitor imatinib mesylate prevented amoeboid movement and markedly reduced homotypic interactions, causing cells to acquire a globular shape to rearrange F-actin to a microvillus form that closely resembled that of CLL cells isolated directly from circulation. CONCLUSION: We suggest that ABL1-induced amoeboid motility and homotypic interaction represent a distinctive early response to the tissue environment by CLL lymphocytes. This response is separate from that induced by chemokine or during heterotypic cell-contact, and may play a role in the initial entry and interactions of CLL lymphocytes in tissues.

Ribosome-associated nucleophosmin 1: increased expression and shuttling activity distinguishes prognostic subtypes in chronic lymphocytic leukaemia.

Two distinct groups of chronic lymphocytic leukaemia (CLL) are distinguished by the presence or absence of somatic hypermutation of the immunoglobulin heavy-chain gene. CLL without somatic hypermutation has an adverse outcome, but the precise biological differences that underlie this more aggressive clinical-course are unclear. Using a proteomic approach, we found that the two prognostic forms of CLL were consistently distinguished according to their protein expression pattern. The most important difference observed related to the different expression of nucleophosmin 1 between the two forms of CLL. This different expression was not related to apoptosis, proliferation or gene mutation. However, co-immunoprecipitation experiments identified an association between nucleophosmin 1 and ribosomal proteins. Using immunocytofluorescence, nucleophosmin 1 expression was identified in the nucleoli and nucleoplasm of all cells, but in a proportion of cells, nucleophosmin had been transferred from the nucleoplasm to the cytoplasm. Both the fluorescent intensity, and the frequency of cytoplasmic nucleophosmin 1 expression, was higher in CLL without somatic hypermutation. We propose therefore, that nucleophosmin 1, in association with ribosomal proteins, undergoes nucleo-cytoplasmic shuttling in CLL. This process is most prominent in un-mutated CLL and may signify altered protein biosynthesis.

Proteomic evaluation of pathways associated with dexamethasone-mediated apoptosis and resistance in multiple myeloma.

We have used global protein expression analysis to characterize the pathways of dexamethasone-mediated apoptosis and resistance in myeloma. Analysis of MM.1S cells by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) identified a series of proteins that were up- and downregulated following dexamethasone treatment. Downregulated proteins included proteins involved in cell survival and proliferation, whereas upregulated proteins were involved in post-translational modification, protein folding and trafficking. A comparison with published gene expression studies identified FK binding protein 5 (FKBP5) (also known as FKBP51), a key regulatory component of the Hsp90-steroid-receptor complex to be increased at the mRNA and protein level postdexamethasone exposure. Quantitative real time polymerase chain reaction and 2D-PAGE analysis of the dexamethasone resistant cell line MM.1R demonstrated no increase in FKBP5, consistent with its association with dexamethasone-mediated apoptosis. Western blot analysis of FKBP5 and other members of the Hsp90-receptor complex showed an increase in FKBP5 whilst FKBP4 (also known as FKBP52) and Hsp90 expression remained constant. No changes were observed in MM.1R. In conclusion, we demonstrated that following steroid receptor signalling, the cell carries out a number of adaptive responses prior to cell death. Interfering with these adaptive responses may enhance the myeloma killing effect of dexamethasone.

Transformed diffuse large B-cell lymphomas with gains of the discontinuous 12q12-14 amplicon display concurrent deregulation of CDK2, CDK4 and GADD153 genes.

Transformation of the indolent follicular lymphoma (FL) to the aggressive diffuse large B-cell lymphoma (DLBCL) results in resistance to therapy with shortened survival. It has been demonstrated that the 12q12-14 region was mainly amplified in DLBCL cases but not in their FL counterparts. Therefore, we examined the DNA copy number and protein expression profiles for CDK2, CDK4 and GADD153, three genes that map to 12q12-14, in a set of 44 paired FL/DLBCL samples from 22 patients. The concordant amplification of these genes occurred in seven of 22 (32%) of FL cases, compared with 15 of 22 (68%) of DLBCL cases. At the protein level, 15 of 22 of the DLBCL samples (68%) showed strong staining for the CDK2 protein, compared with five of 21 of FL samples (24%). The majority of the DLBCL samples (16/22, 72%) expressed the CDK4 protein, whereas the majority of the FL samples (12/21, 57%) showed no expression of this protein. Except for one DLBCL case, no expression of the GADD153 protein could be detected. The deregulation of the CDK2 and CDK4 genes at the genetic and protein levels suggest a functional role for these genes in the transformation process and could potentially provide targets for prognostic tests or therapeutic interventions.

A global expression-based analysis of the consequences of the t(4;14) translocation in myeloma.

PURPOSE: Our purpose in this report was to define genes and pathways dysregulated as a consequence of the t(4;14) in myeloma, and to gain insight into the downstream functional effects that may explain the different prognosis of this subgroup. EXPERIMENTAL DESIGN: Fibroblast growth factor receptor 3 (FGFR3) overexpression, the presence of immunoglobulin heavy chain-multiple myeloma SET domain (IgH-MMSET) fusion products and the identification of t(4;14) breakpoints were determined in a series of myeloma cases. Differentially expressed genes were identified between cases with (n = 5) and without (n = 24) a t(4;14) by using global gene expression analysis. RESULTS: Cases with a t(4;14) have a distinct expression pattern compared with other cases of myeloma. A total of 127 genes were identified as being differentially expressed including MMSET and cyclin D2, which have been previously reported as being associated with this translocation. Other important functional classes of genes include cell signaling, apoptosis and related genes, oncogenes, chromatin structure, and DNA repair genes. Interestingly, 25% of myeloma cases lacking evidence of this translocation had up-regulation of the MMSET transcript to the same level as cases with a translocation. CONCLUSIONS: t(4;14) cases form a distinct subgroup of myeloma cases with a unique gene signature that may account for their poor prognosis. A number of non-t(4;14) cases also express MMSET consistent with this gene playing a role in myeloma pathogenesis.