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.

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.

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.

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.

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.

Nuclear recruitment of A1p145 subunit of replication factor C in the early G1 phase of the cell cycle in Faza 567 hepatoma cell line and hepatocyte primary cultures.

Using a combination of immunoprecipitation and Western blotting with Faza 567 hepatoma cell extracts revealed that the large subunit of replication factor C (A1p145; mRFC140) was in a complex with proliferating cell nuclear antigen (PCNA). Western blotting showed that A1p145 was more abundant in nuclear extracts from butyrate-treated hepatoma cells which blocks the cells in the G1 phase of the cell cycle than from routinely cultured cells. Indirect immunoperoxidase analysis of G1 blocked Faza hepatoma cells localized A1p145 protein predominantly in the nucleoli. When hepatoma cells were stimulated to progress toward the S phase, A1p145 protein was then observed in both the cytoplasm and the nucleoplasm of these cells. Studies with early cultured normal hepatocytes which are progressing from G0 towards G1, also showed a nucleolus distribution for A1p145. This is the first demonstration in mammalian cells that the large subunit of replication factor C is associated with PCNA in the nucleus and that its distribution within cells changes during the cell cycle.

LZIP-1 and LZIP-2: two novel members of the bZIP family.

A large family of bZIP proteins, containing a basic DNA-binding domain and a leucine zipper, have been described that recognize the CRE and AP-1 elements. Here, we have identified two new members, designated LZIP-1 and LZIP-2. The murine cDNA for LZIP-1 coded for a 379-amino-acid (aa) residue protein containing several distinct domains, including a Ser-rich region, a basic DNA-binding region, and an unusually long leucine zipper. A second form, LZIP-2, contained an additional 25 aa in the N-terminal region. Western immunoblotting revealed that antibody raised against part of recombinant LZIP-1 detected both forms in a variety of tissues. Gel mobility shift assays demonstrated that the recombinant protein possessed specific DNA-binding activity for both the CRE AP-1 sites. The present identification of two more ubiquitous members of the bZIP family emphasizes the complex nature of transcription factor interactions at the CRE and AP-1 sites.

Cloning of the large subunit of activator 1 (replication factor C) reveals homology with bacterial DNA ligases.

We have cloned a gene encoding a DNA-binding protein by Southwestern screening of a murine cDNA library with a double-stranded oligonucleotide containing the sequence from the bidirectional promoter of the alpha 1 and alpha 2 collagen IV genes. The middle portion of this 1131-amino acid protein has a region homologous to bacterial DNA ligases, and the more carboxyl portion contains several domains homologous to p40, p38, p37, and p36.5 subunits of activator 1 (A1, also called replication factor C), a human replication protein complex. Western blotting revealed that antiserum generated against part of the recombinant protein reacted specifically with the 145-kDa component of the purified human A1 complex, indicating that it is the murine counterpart of the A1 p145. Characterization of the DNA-binding activity of the recombinant fusion protein by gel mobility-shift assay revealed that it had a preference for a run of pyrimidines on one strand. Deletion analysis using recombinant proteins revealed that the DNA ligase-like domain was required for DNA-binding activity. The finding that the region required for the binding of murine A1 p145 to DNA has similarity to a domain found in DNA ligases suggests that this region may be utilized by both proteins in recognizing DNA.

Altered expression of basement-membrane components and collagenases in ascitic xenografts of OVCAR-3 ovarian cancer cells.

Ascitic ovarian cancer cells, which derive from solid tumors, complicate the treatment of ovarian cancer by spreading throughout the peritoneal cavity. Because basement-membrane components may influence tumor-cell proliferation and dissemination, the present studies examined the production of (a) basement-membrane attachment and migration factors (laminin, fibronectin and type IV collagen); (b) a laminin receptor, the 32/67-kDa laminin-binding protein, the presence of which correlates with malignancy; and (c) metalloproteinases (types I and IV collagenase and stromelysin), by ascitic and cultured OVCAR-3 cells and solid OVCAR-3 tumors. The cultured cells and solid tumors produced high levels of mRNA encoding attachment factors and metalloproteinases, and low levels of mRNA for the 32/67-kDa laminin receptor. In contrast, the ascitic ovarian cells had low or undetectable levels of mRNA encoding laminin, type IV collagen and metalloproteinases, but higher levels of transcripts for the laminin receptor. Our results suggest that the apparent inability of ascitic OVCAR-3 cells to attach to host-tissue surfaces may be a consequence, in part, of low levels of expression of laminin, type IV collagen and/or type IV collagenase.

A novel sequence in the type IV collagen promoter binds nuclear proteins from Engelbreth-Holm-Swarm tumor.

The production of extracellular matrix proteins is an important element of tumor formation, and alterations in matrix protein metabolism may be critical to the process of tumor metastasis. Abundant expression of type IV collagen, the major structural protein of the basement membrane, is characteristic of the Engelbreth-Holm-Swarm (EHS) mouse sarcoma. In the present study, we evaluated mechanisms of transcriptional regulation of type IV collagen genes by analysing nuclear factors that bind to the promoter region. Gel mobility-shift assays indicated that specific proteins from EHS tumor bound the promoter and generated several unique shift patterns. The specific sequences to which these proteins bound were determined using DNAase I protection assays. DNA-binding proteins protected two regions from DNAase I digestion. The first region was similar to a GC box, the binding site for the transcription factor Sp1. The other footprint was a 30-bp region that contained the novel sequence motif, 'CCCTCCC' present in several other extracellular matrix promoters. Nuclear extracts isolated from tissues that variably express type IV collagen bound to this protected sequence with distinctly different shift patterns. Furthermore, in highly expressing tissues, unlabeled oligonucleotides containing the 'CCCTCCC' motif effectively inhibited nuclear protein binding with the entire promoter. Thus, it is likely that a novel protein or protein complex binds to these sequences. Furthermore, these sequences appear to be unique to the genes that encode basement membrane proteins, suggesting a specific role in their regulation.

Activation of collagen IV gene expression in F9 teratocarcinoma cells by 3-deazaadenosine analogs. Indirect inhibitors of methylation.

3-Deazaadenosine analogs can function as inhibitors and also as alternative substrates of S-adenosylhomocysteine (AdoHcy) hydrolase. In cells treated with the analogs, AdoHcy invariably accumulates, leading to inhibition of cellular methylation. F9 teratocarcinoma cells, stably transfected with two collagen (IV) promoter-enhancer-CAT constructs and treated with 10 microM 3-deazaadenosine, 3-deaza-(+-)-aristeromycin or 3-deazaneplanocin, showed a strong induction of CAT activities without affecting differentiation. In comparison, the same 3-deaza analogs did not affect the CAT activity in F9 cells transfected with the beta-actin promoter-CAT construct. Furthermore, Northern blot analysis of endogenous mRNA from wild-type F9 cells treated with the 3-deaza nucleosides all showed an induction of the collagen alpha 1(IV) chain mRNA. Thus, the 3-deaza analogs most likely affect DNA methylation because their results are consistent with the previous observation that the integrated collagen alpha 1(IV) promoter-enhancer constructs were activated with 5-azacytidine.

Characterization of a cis-acting element required for efficient transcriptional activation of the collagen IV enhancer.

Two regulatory regions in the murine collagen IV enhancer were identified. Transient transfection assays delimited a 210-base pair fragment within the first intron of the alpha 1(IV) collagen gene that had significant transcriptional enhancer activity. DNase I protection and gel mobility shift confirmed that two regions, designated footprints A and B, within this fragment bound nuclear factors. Gel shift studies suggested that the CCTTATCTCTGATGG motif (A-34) in the footprint A region was important for specific nuclear factor binding. Mutations in the A-34 motif abolished factor binding as detected by gel shift and resulted in a significant decrease in enhancer activity in transient transfection assays of F9 teratocarcinoma cells. Two putative transcription factors of Mr = 37,000 and Mr = 94,000, which interact with the A-34 motif, were purified from Engelbreth-Holm-Swarm tumor tissue using DEAE-Sephacel, heparin-Sepharose, salmon sperm DNA-Sepharose, and specific A-34 oligonucleotide affinity chromatography. Southwestern analysis revealed that both of these factors were capable of binding the A-34 oligonucleotide directly and did not require additional subunits for binding. These data suggest that positively acting transcription factor(s) interact with the A-34 site in the enhancer and are required for efficient transcription of the alpha 1 and alpha 2(IV) collagen chain genes.