Production and persistence of specific antibodies in COVID-19 patients with hematologic malignancies: role of rituximab.

The ability of patients with hematologic malignancies (HM) to develop an effective humoral immune response after COVID-19 is unknown. A prospective study was performed to monitor the immune response to SARS-CoV-2 of patients with follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), chronic lymphoproliferative disorders (CLD), multiple myeloma (MM), or myelodysplastic/myeloproliferative syndromes (MDS/MPN). Antibody (Ab) levels to the SARS-CoV-2 nucleocapsid (N) and spike (S) protein were measured at +1, +3, +6 months after nasal swabs became PCR-negative. Forty-five patients (9 FL, 8 DLBCL, 8 CLD, 10 MM, 10 MDS/MPS) and 18 controls were studied. Mean anti-N and anti-S-Ab levels were similar between HM patients and controls, and shared the same behavior, with anti-N Ab levels declining at +6 months and anti-S-Ab remaining stable. Seroconversion rates were lower in HM patients than in controls. In lymphoma patients mean Ab levels and seroconversion rates were lower than in other HM patients, primarily because all nine patients who had received rituximab within 6 months before COVID-19 failed to produce anti-N and anti-S-Ab. Only one patient requiring hematological treatment after COVID-19 lost seropositivity after 6 months. No reinfections were observed. These results may inform vaccination policies and clinical management of HM patients.

Profiling Autoantibodies against Salivary Proteins in Sicca Conditions.

Salivary gland dysfunction occurs in several autoimmune and immune-related conditions, including Sjögren syndrome (SS); immune checkpoint inhibitor-induced sicca (ICIS) that develops in some cancer patients and is characterized by severe, sudden-onset dry mouth; and autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Although subjects with these conditions present with oral dryness and often exhibit inflammatory infiltration of the salivary gland, little is known about the B-cell humoral responses directed against salivary gland protein targets. In this study, autoantibodies were evaluated against Ro52, Ro60, and La, as well as against a panel of 22 proteins derived from the salivary proteome. The tested cohort included healthy volunteers and subjects with SS, ICIS, and APECED without and with sicca. As expected, a high percentage of autoantibody seropositivity was detected against Ro52, Ro60, and La in SS, but only a few ICIS patients were seropositive for these autoantigens. A few APECED subjects also harbored autoantibodies to Ro52 and La, but only Ro60 autoantibodies were weakly associated with a small subset of APECED patients with sicca. Additional testing of the salivary panel failed to detect seropositive autoantibodies against any of the salivary-enriched proteins in the SS and ICIS subjects. However, APECED subjects selectively demonstrated seropositivity against BPI fold containing family A member 1 (BPIFA1), BPI fold containing family A member 2 (BPIFA2)/parotid salivary protein (PSP), and lactoperoxidase, 3 salivary-enriched proteins. Moreover, high levels of serum autoantibodies against BPIFA1 and BPIFA2/PSP occurred in 30% and 67% of the APECED patients with sicca symptoms, respectively, and were associated with an earlier age onset of oral dryness (P = 0.001). These findings highlight the complexity of humoral responses in different sicca diseases and provide new insights and biomarkers for APECED-associated sicca (ClinicalTrials.gov: NCT00001196; NCT00001390; NCT01425892; NCT01386437).

Targeted TNF-α Overexpression Drives Salivary Gland Inflammation.

Chronic inflammation of the salivary glands from pathologic conditions such as Sjögren's syndrome can result in glandular destruction and hyposalivation. To understand which molecular factors may play a role in clinical cases of salivary gland hypofunction, we developed an aquaporin 5 (AQP5) Cre mouse line to produce genetic recombination predominantly within the acinar cells of the glands. We then bred these mice with the TNF-αglo transgenic line to develop a mouse model with salivary gland-specific overexpression of TNF-α; which replicates conditions seen in sialadenitis, an inflammation of the salivary glands resulting from infection or autoimmune disorders such as Sjögren's syndrome. The resulting AQP5-Cre/TNF-αglo mice display severe inflammation in the salivary glands with acinar cell atrophy, fibrosis, and dilation of the ducts. AQP5 expression was reduced in the salivary glands, while tight junction integrity appeared to be disrupted. The immune dysregulation in the salivary gland of these mice led to hyposalivation and masticatory dysfunction.

New technologies for studying the complexity of oral diseases.

Several new technologies are providing useful diagnostic tools and new information related to the pathogenesis of certain oral diseases. In this review, we describe several of these technologies including gene and microRNA arrays, proteomics, and antigen arrays as they relate to the study of Sjögren's syndrome and head and neck cancer. A common theme is the systematic analysis of large-scale inventories of RNAs, proteins, and autoantibody biomarkers revealing information not previously recognized. We also discuss metagenomic approaches that characterize the many different microorganisms present in the oral cavity that may impact oral and human health. Lastly, we describe applications of a new type of antibody-profiling technology termed Luciferase Immunoprecipitation Systems (LIPS), which has a wide dynamic range of detection of both linear and conformational epitopes needed for optimum diagnostics and biomarker discovery. We propose that the information offered by these technologies will enhance our ability to diagnose, treat, and further understand the pathogenesis of multiple oral diseases.

Salivary anti-Ro60 and anti-Ro52 antibody profiles to diagnose Sjogren's Syndrome.

Simple and non-invasive saliva-based diagnostics may be useful for the identification, understanding, and monitoring of autoimmune and infectious diseases. Previously, Luciferase Immunoprecipitation Systems (LIPS) were used for sensitive detection of patient serum autoantibodies in Sjögren's Syndrome (SjS), a chronic autoimmune disease affecting the salivary and lacrimal glands. Here we explored the ability of LIPS to diagnose SjS based on IgG autoantibodies in patient saliva. From LIPS testing, anti-Ro60 autoantibodies were detected in the saliva of 70% (19/27) of SjS patients with 96% specificity. Positive anti-Ro60 autoantibodies were also found in 70% of the matched serum samples (96% specificity). LIPS detected Ro52 autoantibodies in the saliva and serum of 67% of SjS patients with 100% specificity. Overall, the autoantibody titers in saliva were approximately 4000-fold lower by volume than serum, but still distinguished seropositive patients from controls. These results suggest that LIPS salivary-based testing for SjS autoantibodies is a practical alternative to serum and compatible with point-of-care testing.

The genomic structure of the human SPEC1 gene reveals complex splicing and close promoter proximity to the AF1q translocation gene.

SPECs are small Cdc42 signaling molecules. In mammals, two genes, SPEC1 and SPEC2, encode proteins of 79 and 84 amino acid residues, respectively. Here we report the expression and genomic organization of the human SPEC1 gene. Using Northern blot analysis, three major SPEC1 mRNA transcripts of 1.6, 3.3, and 6.3 kb were detected. Identification and sequencing of different sized SPEC1 cDNA clones revealed that the transcript size heterogeneity was due to alternative splicing in the 3'-untranslated region. In addition, a distinct SPEC1 splice variant from within the coding sequence, SPEC1-beta, was identified and detected in a variety of human tissues. Analysis of the genomic organization of SPEC1 revealed that the coding sequence of the SPEC1 isoform was derived from exons 2, 3 and 4, while the SPEC1-beta isoform was derived from exon 2 and a read-through event of intron 2. Examination of the 5'-end of the SPEC1 genomic sequence revealed that AF1q, a previously identified gene involved in translocations with the MLL (mixed-lineage leukemia) gene, was 631 bp away in a head-to-head orientation. This intergenic sequence containing the putative promoter region for both SPEC1 and AF1q genes did not contain a TATA box or CAAT box. Transfection experiments using an AF1q promoter luciferase reporter construct in a variety of cells including Cos1 cells, Jurkat T-cells, MCF-7 breast cancer cells, and NIH-3T3 fibroblasts showed no promoter activity. In contrast, a SPEC1 promoter luciferase reporter construct showed high levels of reporter activity in Cos1 and MCF-7 cells, low activity in NIH-3T3 fibroblasts and no activity in Jurkat T-cells. These promoter analyses suggest that although SPEC1 and AF1q genes share the same promoter region, they are not coordinately regulated.

Evolutionary expansion of CRIB-containing Cdc42 effector proteins.

Cdc42, a small GTPase, regulates actin polymerization and other signaling pathways through interaction with many different downstream effector proteins. Most of these effector proteins contain a Cdc42-binding domain, called a CRIB domain. Here, we describe the evolutionary analysis of these CRIB-containing proteins in yeast, worms, flies and humans. The number of CRIB-containing effector proteins increases from yeast to humans, involving both an increase within families and the emergence of new families. These evolutionary changes correlate with the development of the more complex signaling pathways present in higher organisms.

A new family of Cdc42 effector proteins, CEPs, function in fibroblast and epithelial cell shape changes.

Cdc42, a Rho GTPase, regulates the organization of the actin cytoskeleton by its interaction with several distinct families of downstream effector proteins. Here, we report the identification of four new Cdc42-binding proteins that, along with MSE55, constitute a new family of effector proteins. These molecules, designated CEPs, contain three regions of homology, including a Cdc42 binding domain and two unique domains called CI and CII. Experimentally, we have verified that CEP2 and CEP5 bind Cdc42. Expression of CEP2, CEP3, CEP4, and CEP5 in NIH-3T3 fibroblasts induced pseudopodia formation. Fibroblasts coexpressing dominant negative Cdc42 with CEP2 or expressing a Cdc42/Rac interactive binding domain mutant of CEP2 did not induce pseudopodia formation. In primary keratinocytes, CEP2- and CEP5-expressing cells showed reduced F-actin localization at the adherens junctions with an increase in thin stress fibers that extended the length of the cell body. Keratinocytes expressing CEPs also showed an altered vinculin distribution and a loss of E-cadherin from adherens junctions. Similar effects were observed in keratinocytes expressing constitutively active Cdc42, but were not seen with a Cdc42/Rac interactive binding domain mutant of CEP2. These results suggest that CEPs act downstream of Cdc42 to induce actin filament assembly leading to cell shape changes.

SPECs, small binding proteins for Cdc42.

The Rho GTPase, Cdc42, regulates a wide variety of cellular activities including actin polymerization, focal complex assembly, and kinase signaling. We have identified a new family of very small Cdc42-binding proteins, designated SPECs (for Small Protein Effector of Cdc42), that modulates these regulatory activities. The two human members, SPEC1 and SPEC2, encode proteins of 79 and 84 amino acids, respectively. Both contain a conserved N-terminal region and a centrally located CRIB (Cdc42/Rac Interactive Binding) domain. Using a yeast two-hybrid system, we found that both SPECs interact strongly with Cdc42, weakly with Rac1, and not at all with RhoA. Transfection analysis revealed that SPEC1 inhibited Cdc42-induced c-Jun N-terminal kinase (JNK) activation in COS1 cells in a manner that required an intact CRIB domain. Immunofluorescence experiments in NIH-3T3 fibroblasts demonstrated that both SPEC1 and SPEC2 showed a cortical localization and induced the formation of cell surface membrane blebs, which was not dependent on Cdc42 activity. Cotransfection experiments demonstrated that SPEC1 altered Cdc42-induced cell shape changes both in COS1 cells and in NIH-3T3 fibroblasts and that this alteration required an intact CRIB domain. These results suggest that SPECs act as novel scaffold molecules to coordinate and/or mediate Cdc42 signaling activities.

Identification of syntenin as a protein of the apical early endocytic compartment in Madin-Darby canine kidney cells.

We used flow cytometry to sort and analyze apical and basolateral endocytic vesicles from filter-grown Madin-Darby canine kidney (MDCK) cells after membrane internalization of the lipophilic fluorescent probe trimethylamino-diphenylhexatriene. Western blot analysis of sorted fractions showed enrichment of the early endosomal markers transferrin receptor and the small GTPase Rab5. Two-dimensional gel analysis indicated that the apical and basolateral early endosomes differed significantly in their protein composition. We found nine polypeptides to be specifically enriched in apical or basolateral endocytic vesicles. An apical protein identified by microsequencing was the adaptor molecule syntenin. This protein contains two PDZ domains (PSD-95, Dlg, and ZO-1 homology) that bind syndecan and ephrin-B2 cytoplasmic domains. In MDCK cells, transiently overexpressed Myc-tagged syntenin localized to both plasma membrane domains and to an intracellular vesicular compartment. Syntenin positive vesicles colocalized with internalized transferrin in the perinuclear region. In addition, syntenin colocalized in the apical supranuclear region with Rab5 and Rab11; the latter is a marker for the apical recycling endosomes in MDCK cells.

MSE55, a Cdc42 effector protein, induces long cellular extensions in fibroblasts.

Cdc42 is a member of the Rho GTPase family that regulates multiple cellular activities, including actin polymerization, kinase-signaling activation, and cell polarization. MSE55 is a nonkinase CRIB (Cdc42/Rac interactive-binding) domain-containing molecule of unknown function. Using glutathione S-transferase-capture experiments, we show that MSE55 binds to Cdc42 in a GTP-dependent manner. MSE55 binding to Cdc42 required an intact CRIB domain, because a MSE55 CRIB domain mutant no longer interacted with Cdc42. To study the function of MSE55 we transfected either wild-type MSE55 or a MSE55 CRIB mutant into mammalian cells. In Cos-7 cells, wild-type MSE55 localized at membrane ruffles and increased membrane actin polymerization, whereas expression of the MSE55 CRIB mutant showed fewer membrane ruffles. In contrast to these results, MSE55 induced the formation of long, actin-based protrusions in NIH 3T3 cells as detected by immunofluorescence and live-cell video microscopy. MSE55-induced protrusion formation was blocked by expression of dominant-negative N17Cdc42, but not by expression of dominant-negative N17Rac. These findings indicate that MSE55 is a Cdc42 effector protein that mediates actin cytoskeleton reorganization at the plasma membrane.

Cloning, central nervous system expression and chromosomal mapping of the mouse PAK-1 and PAK-3 genes.

Two cDNAs encoding PAK kinases were isolated from a mouse embryo library by screening with a PCR-generated probe derived from the kinase domain of a rat PAK kinase. These cDNAs, designated PAK-1 and PAK-3, encode mouse PAK kinases of 545 and 544 amino acids, respectively. Both proteins possess an N-terminal Cdc42/Rac interacting binding domain (CRIB) and a C-terminal serine/threonine kinase domain. Comparison of the two mouse PAK kinases revealed that the proteins show 87% amino acid identity. Northern analysis of a multiple mouse tissue blot with a PAK-1 probe detected a 3.0kb transcript that was almost exclusively expressed in the brain and spinal cord compared to other tissues such as lung, liver and kidney. A similar pattern of central nervous system tissue expression of PAK-3 transcripts of 3.6 and 8kb was also observed. Analysis of two multilocus genetic crosses localized Pak1 and Pak3 to a position on chromosome 7 and X, respectively. The high level of PAK-1 and PAK-3 kinase expression in the mouse brain and spinal cord suggests a potentially important role for these kinases in the control of the cellular architecture and/or signaling in the central nervous system.

Cloning, genomic organization and chromosomal assignment of the mouse p190-B gene.

The p190 family of GTPases consists of at least two different isoforms both containing an N-terminal GTPase and a C-terminal Rho GAP domain. Here we have isolated and characterized genomic and cDNA clones spanning the entire coding region of the mouse p190-B gene. Genomic data were obtained by sequencing plasmid subclones of two overlapping mouse genomic phage clones. Interestingly, a single 3.9 kb exon was found to contain approx. 80% of the coding region of the mouse p190-B protein (amino acid residues 1-1238) including the 5'-untranslated region, the N-terminal GTPase domain and a middle domain of unknown function. Missing from this exon, however, was the C-terminal Rho GAP domain, which was cloned from mouse brain mRNA using reverse transcriptase polymerase chain reaction. Comparison of the mouse with the human p190-B proteins revealed that approx. 97% of the amino acid residues were identical. Northern analysis of total RNA from a variety of mouse tissues detected ubiquitous expression of two p190-B transcripts of 4.0 and 6.8 kb in size. Analysis of two multilocus genetic crosses localized the mouse gene, Gfi2, to a position on chromosome 12, consistent with the mapping of the human gene to a position of conserved synteny on chromosome 14. The high level of sequence homology between the human and the mouse suggests that there is a strong selective pressure to maintain the p190-B protein structure.

Formation of recombinant triple-helical [alpha 1(IV)]2 alpha 2(IV) collagen molecules in CHO cells.

Collagen IV molecules represent a major structural component of basement membranes providing a network of support for the supramolecular structure. Like other collagens, collagen IV forms a triple-helical molecule composed of three alpha chains. Six different alpha chains exist for collagen IV, although the most common isoform consists of two alpha 1(IV) and one alpha 2(IV) chain. To understand the molecular mechanism of triple-helical formation of collagen IV, we expressed recombinant alpha 1(IV) and alpha 2(IV) mouse collagen chains in Chinese hamster ovary (CHO) cells. An expression vector containing the full length cDNA for the mouse alpha 1(IV) chain was stably transfected into CHO cells and a cell line, A222, which expressed recombinant alpha 1(IV) chains was selected. These A222 cells were then infected with a retroviral expression vector containing the mouse alpha 2(IV) chain and a cell line, A222-A2, stably expressing both recombinant alpha 1(IV) and alpha 2(IV) chains was obtained. Immunoprecipitation of A222 cell lysates revealed a high level of alpha 1(IV) chain monomer, which was unable to form a homotrimer. Analysis of A222-A2 cell lysates revealed the presence of both monomeric alpha 2(IV) and alpha 1(IV) chains as well as a higher molecular weight collagen IV species. Second dimensional SDS-PAGE analysis demonstrated that the high molecular weight species was a heterotrimer consisting of two alpha 1(IV) and one alpha 2(IV) chain. This heterotrimer collagen IV species was pepsin-resistant indicating the formation of a stable triple-helical structure. Pulse-chase experiments showed that the monomer alpha 1(IV) chain was secreted, but at a much slower rate than the heterotrimer. Together these results demonstrate that the alpha 1(IV) chain is not capable of forming homotrimers and suggest that the coexpression with the alpha 2(IV) chain is necessary to form a triple-helical structure.

Rac and Cdc42 induce actin polymerization and G1 cell cycle progression independently of p65PAK and the JNK/SAPK MAP kinase cascade.

Rac and Cdc42 regulate a variety of responses in mammalian cells including formation of lamellipodia and filopodia, activation of the JNK MAP kinase cascade, and induction of G1 cell cycle progression. Rac is also one of the downstream targets required for Ras-induced malignant transformation. Rac and Cdc42 containing a Y40C effector site substitution no longer intact with the Ser/Thr kinase p65PAK and are unable to activate the JNK MAP kinase pathway. However, they still induce cytoskeletal changes and G1 cell cycle progression. Rac containing an F37A effector site substitution, on the other hand, no longer interacts with the Ser/Thr kinase p160ROCK and is unable to induce lamellipodia or G1 progression. We conclude that Rac and Cdc42 control MAP kinase pathways and actin cytoskeleton organization independently through distinct downstream targets.

Expression of laminin gamma 1 cultured hepatocytes involves repeated CTC and GC elements in the LAMC1 promoter.

Laminin gamma 1 chain is present in all basement membranes and is expressed at high levels in various diseases, such as hepatic fibrosis. We have identified cis- and trans-acting elements involved in the regulation of this gene in normal rat liver, as well as in hepatocyte primary cultures and hepatoma cell lines. Northern-blot analyses showed that laminin gamma 1 mRNA was barely detectable in freshly isolated hepatocytes and expressed at high levels in hepatocyte primary cultures, as early as 4 h after liver dissociation. Actinomycin D and cycloheximide treatment in vivo and in vitro indicated that laminin gamma 1 overexpression in cultured hepatocytes was under the control of transcriptional mechanisms. Transfection of deletion mutants of the 5' flanking region of murine LAMC1 gene in hepatoma cells that constitutively express laminin gamma 1 indicated that regulatory elements were located between -594 bp and -94 bp. This segment included GC- and CTC-containing motifs. Gel-shift analyses showed that two complexes were resolved with different affinity for the CTC sequence depending on the location of the GC box. The pattern of complex formation with nuclear factors from freshly isolated and cultured hepatocytes was different from that obtained with total liver and similar to that with hepatoma cells. Southwestern analysis indicated that several polypeptides bound the CTC-rich sequence. Affinity chromatography demonstrated that A M(r) 60,000 polypeptide was a major protein binding to the CTC motif. This polypeptide is probably involved in the transcriptional activation of various proto-oncogenes and extracellular matrix genes that are expressed at high levels in both hepatoma cells and early hepatocyte cultures.

p190-B, a new member of the Rho GAP family, and Rho are induced to cluster after integrin cross-linking.

p120GAP forms distinct complexes with two phosphoproteins, p62 and p190. Here we have cloned a cDNA encoding a protein with 51% amino acid identity to p190 (hereafter designated p190-A) and have designated it p190-B. The N-terminal portion of p190-B contained several motifs characteristic of a GTPase domain, while its C terminus contained a Rho GAP domain. A recombinant Rho GAP domain polypeptide showed GAP activity for RhoA, Rac1, and G25K/CDC42Hs. Immunoprecipitation and immunofluorescence studies demonstrated that p190-B protein was expressed in a variety of cells and was localized diffusely in the cytoplasm and in fibrillar patterns that co-localized with the alpha 5 beta 1 integrin receptor for fibronectin. Adhesion of fibronectin-coated latex beads to cells resulted in recruitment of significant amounts of p190-B and Rho to the plasma membrane beneath the site of bead binding. In contrast, beads coated with polylysine or concanavalin A were unable to recruit p190-B or Rho. Additionally, anti-beta 1 or anti-alpha 5 integrin antibody-coated beads were also able to recruit large amounts of p190-B and Rho. These results identify a novel second member of the p190 family and establish the existence of a novel transmembrane link between integrins and a new protein p190-B and Rho.

Matrigel induces thymosin beta 4 gene in differentiating endothelial cells.

We performed differential cDNA hybridization using RNA from endothelial cells cultured for 4 hours on either plastic or basement membrane matrix (Matrigel), and identified early genes induced during the morphological differentiation into capillary-like tubes. The mRNA for one clone, thymosin beta 4, was increased 5-fold. Immunostaining localized thymosin beta 4 in vivo in both growing and mature vessels as well as in other tissues. Endothelial cells transfected with thymosin beta 4 showed an increased rate of attachment and spreading on matrix components, and an accelerated rate of tube formation on Matrigel. An antisense oligo to thymosin beta 4 inhibited tube formation on Matrigel. The results suggest that thymosin beta 4 is induced and likely involved in differentiating endothelial cells. Thymosin beta 4 may play a role in vessel formation in vivo.

A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases.

Rho, Rac, and Cdc42 are small GTPases that regulate the formation of a variety of actin structures and the assembly of associated integrin complexes, but little is known about the target proteins that mediate their effects. Here we have used a motif-based search method to identify putative effector proteins for Rac and Cdc42. A search of the GenBankTM data base for similarity with the minimum Cdc42/Rac interactive binding (CRIB) region of a potential effector protein p65PAK has identified over 25 proteins containing a similar motif from a range of different species. These candidate Cdc42/Rac-binding proteins include family members of the mixed lineage kinases (MLK), a novel tyrosine kinase from Drosophila melanogaster (DPR2), a human protein MSE55, and several novel yeast and Caenorhabditis elegans proteins. Two murine p65PAK isoforms and a candidate protein from C. elegans, F09F7.5, interact strongly with the GTP form of both Cdc42 and Rac, but not Rho in a filter binding assay. Three additional candidate proteins, DPR2, MSE55, and MLK3 showed binding to the GTP form of Cdc42 and weaker binding with Rac, and again no interaction with Rho. These results indicate that proteins containing the CRIB motif bind to Cdc42 and/or Rac in a GTP-dependent manner, and they may, therefore, participate in downstream signaling.