Juan R. de los Toyos

Protection against Pneumococcal Pneumonia in Mice by Monoclonal Antibodies to Pneumolysin

ABSTRACT Pneumolysin (PLY) is an important virulence factor of Streptococcus pneumoniae. We examined the ability of three murine monoclonal antibodies (MAbs) to PLY (PLY-4, PLY-5, and PLY-7) to affect the course of pneumococcal pneumonia in mice. The intravenous administration of antibodies PLY-4 and PLY-7 protected the mice from the lethal effect of the purified toxin. Mice treated with PLY-4 before intranasal inoculation of S. pneumoniae type 2 survived longer (median survival time, 100 h) than did untreated animals (median survival time, 60 h) (P < 0.0001). The median survival time for mice treated with a combination of PLY-4 and PLY-7 was 130 h, significantly longer than that for mice given isotype-matched indifferent MAbs (P = 0.0288) or nontreated mice (P = 0.0002). The median survival time for mice treated with a combination of three MAbs was significantly longer (>480 h) than that for mice treated with PLY-5 (48 h; P < 0.0001), PLY-7 (78 h; P = 0.0007), or PLY-4 (100 h; P = 0.0443) alone. Similarly, the survival rate for mice treated with three MAbs (10 of 20 mice) was significantly higher than the survival rate obtained with PLY-5 (1 of 20; P = 0.0033), PLY-4 (2 of 20; P = 0.0138), or PLY-7 (3 of 20; P = 0.0407) alone. These results suggest that anti-PLY MAbs act with a synergistic effect. Furthermore, MAb administration was associated with a significant decrease in bacterial lung colonization and lower frequencies of bacteremia and tissue injury with respect to the results for the control groups.

The conserved undecapeptide shared by thiol‐activated cytolysins is involved in membrane binding

Thiol‐activated cytolysins share a conserved hydrophobic, Trp‐rich undecapeptide that is suggested to be involved in membrane binding and intercalation. The neutralizing monoclonal antibody PLY‐5 recognizes all members of this toxin family and peptide mapping assigned its epitope to the undecapeptide motif. This antibody inhibited binding of the toxins to host cell membranes and the epitope was no longer available for binding when a preformed toxin/membrane complex was tested. These results confirm the model of cytolysin binding suggested by structural data.

A specific and ultrasensitive chemiluminescent sandwich ELISA test for the detection and quantitation of pneumolysin.

A chemiluminescent sandwich ELISA test has been developed for the detection and quantitation of pneumolysin. The test is based on a mouse monoclonal as the capture antibody and on rabbit polyclonal IgGs as detection antibodies, in combination with an anti-rabbit IgG alkaline phosphatase conjugate. The estimated detection limit of the purified recombinant toxin in phosphate-buffered saline with 0.05% Triton X-100 is around 5 pg ml−1, with averaged intra- and inter-assay variation coefficients of 7% and 13.5%, respectively. The assay has been applied to the quantitation of pneumolysin in pneumococcal isolates, providing, for the first time, a direct measurement of the amount of the toxin produced by different strains; a variation has been found in their pneumolysin content. The test is highly specific as no other purified toxins or human pneumonia- or meningitis-associated bacteria yielded false-positive results. This specific and highly sensitive method could help in the diagnosis of human infections.

Expression of ERp5 and GRP78 on the membrane of chronic lymphocytic leukemia cells: association with soluble MICA shedding

MICA is a ligand of the activating receptor NKG2D, expressed by NK and T cells. MICA expression is induced in cancer cells favoring their elimination by the immune system; however, many advanced tumors shed soluble MICA (sMICA), which impairs NKG2D-mediated cytotoxicity. ERp5 and GRP78 are endoplasmic reticulum-resident proteins that are translocated to the surface of epithelial tumor cells where they interact with MICA and are involved in sMICA shedding. In this study, we analyze the role of ERp5 and GRP78 in sMICA shedding in chronic lymphocytic leukemia (CLL). Immunofluorescence and flow cytometry analyses showed that ERp5 and GRP78 were significantly expressed on the surface of B cells and leukemia cells, but they were not expressed on T cells. The expression of ERp5 and GRP78 was significantly higher in leukemia cells than in B cells from controls. ERp5 and GRP78 co-localized with MICA on the surface of leukemia cells and the levels of expression of ERp5 and GRP78 correlated with the level of expression of membrane-bound MICA in CLL patients. Associated with higher expression of membrane-bound ERp5 and GRP78, serum sMICA levels were approximately threefold higher in patients than in controls. Elevated sMICA levels in CLL patients were associated with the down-modulation of NKG2D surface expression on CD8 T cells. Finally, pharmacological inhibition of B cell lines and stimulated leukemia cells showed that ERp5 activity is involved in sMICA shedding in CLL. In conclusion, these results uncover a molecular mechanism which regulates MICA protein shedding and immune evasion in CLL.

The innate immunity role of cathepsin-D is linked to Trp-491 and Trp-492 residues of listeriolysin O

Listeriolysin O (LLO) is a thiol‐activated cytolysin secreted by Listeria monocytogenes. LLO and phosphatidylinositol phospholipase C are two essential virulence factors, which this bacterium needs to escape from the phagosomal compartment to the cytoplasm. Cathepsin‐D specifically cleaves LLO, between the Trp‐491 (tryptophan amino acid in three letter nomenclature) and Trp‐492 residues of the conserved undecapeptide sequence, ECTGLAWEWWR, in the domain 4 of LLO (D4). Moreover, these residues also correspond to the phagosomal‐binding epitope. Cathepsin‐D had no effect on phosphatidylinositol phospholipase C. We have observed that cathepsin‐D cleaved the related cholesterol‐dependent cytolysin pneumolysin at the same undecapeptide sequence between Trp‐435 and Trp‐436 residues. These studies also revealed an additional cathepsin‐D cleavage site in the pneumolysin D4 domain localized in the 361‐GDLLLD‐366 sequence. These differences might confer a pathogenic advantage to listeriolysin O, increasing its resistance to phagosomal cathepsin‐D action by reducing the number of cleavages sites in the D4 domain. Using ΔLLO/W491A and ΔLLO/W492A bacterial mutants, we reveal that the Trp‐491 residue has an important role linked to cathepsin‐D in Listeria innate immunity.

Characterisation of mouse monoclonal antibodies for pneumolysin: fine epitope mapping and V gene usage

Pneumolysin (PLY) is a cholesterol-dependent cytolysin (CDC) produced by Streptococcus pneumoniae, the main cause of community-acquired pneumonia. We have applied a set of diverse molecular methodologies (PCR-derived PLY peptides, biopanning of a library of phage-displayed random nonapeptides, indirect ELISA and competition tests with soluble peptides) to achieve concordant complementary observations in order to obtain a fine epitope mapping of three mouse monoclonal antibodies (PLY-4, PLY-7 and PLY-8) for PLY. PLY-4 seems to recognise a conformation-dependent epitope with a core reactivity involving R232. The epitopes recognised by PLY-7 and PLY-8 are within the sequences (401)GQDLTAH(407) and (450)KRTISIWGT(458), respectively. PLY-7 also recognises suilysin (SLY), in which the homologous reactive amino acid stretch is (429)GVNLTSH(435). In a homology model of PLY with the crystal structure of perfringolysin O (PFO), R232 is part of a well-exposed contorted loop on the edge of the concave and convex faces of domain 1. The sequences reactive with PLY-7 and PLY-8 would conform one of the loops at the bottom of domain 4 and a beta strand of one of the two beta sheets of this domain, respectively. Western blot analyses carried out with anti-PLY rabbit IgG and polyclonal mouse serum identified stretches comprising residues 40-98, 199-248, 352-414 and 415-471 of PLY as immunogenic and antigenic; altogether with their recognition by the monoclonal antibodies herein considered, these results stress the immunological significance of domains 1 and 4 of the PLY molecule. PLY-4, PLY-7 and PLY-8 share the same Vkappa chain; this chain and that of the PLY-5 monoclonal antibody are essentially in germline configuration, whereas the VH regions of these monoclonals come from diverse gene segments and are mutated.

COL11A1/(pro)collagen 11A1 expression is a remarkable biomarker of human invasive carcinoma-associated stromal cells and carcinoma progression.

The COL11A1 human gene codes for the α1 chain of procollagen 11A1 and mature collagen 11A1, an extracellular minor fibrillar collagen. Under regular conditions, this gene and its derived products are mainly expressed by chondrocytes and mesenchymal stem cells as well as osteoblasts. Normal epithelial cells and quiescent fibroblasts from diverse locations do not express them. Mesenchyme-derived tumors and related conditions, such as scleroderma and keloids, are positive for COL11A1/(pro)collagen 11A1 expression, as well as high-grade human gliomas/glioblastomas. This expression is almost absent in benign pathological processes such as breast hyperplasia, sclerosing adenosis, idiopathic pulmonary fibrosis, cirrhosis, pancreatitis, diverticulitis, and inflammatory bowel disease. By contrast, COL11A1/(pro)collagen 11A1 is highly expressed by activated stromal cells of the desmoplastic reaction of different human invasive carcinomas, and this expression is correlated with carcinoma aggressiveness and progression, and lymph node metastasis. COL11A1 upregulation has been shown to be associated to TGF-β1, Wnt, and Hh signaling pathways, which are especially active in cancer-associated stromal cells. At the front of invasive carcinomas, neoplastic epithelial cells, putatively undergoing epithelial-to-mesenchymal transition, and carcinoma-derived cells with highly metastatic capabilities, can express COL11A1. Thus, in established metastases, the expression of COL11A1/(pro)collagen 11A1 could rely on both the metastatic epithelial cells and/or the accompanying activated stromal cells. COL11A1/(pro)collagen 11A1 expression is a remarkable biomarker of human carcinoma-associated stromal cells and carcinoma progression.

Overexpression of COL11A1 by Cancer-Associated Fibroblasts: Clinical Relevance of a Stromal Marker in Pancreatic Cancer

Background The collagen11A1 (COL11A1) gene is overexpressed in pancreatic cancer. The expression of COL11A1 protein could be involved in desmoplastic events in pancreatic cancer, but an antibody that specifically stains the COL11A1 protein is not currently available. Methods and findings A total of 54 pancreatic ductal adenocarcinomas (PDAC), 23 chronic pancreatitis (CP) samples, and cultured peritumoral stromal cells of PDAC (passages 3-6) were studied. Normal human pancreas tissue samples were obtained through a cadaveric organ donation program. 1) Validation of COL11A1 gene overexpression by q-RT-PCR. Findings: the expression of COL11A1 gene is significantly increased in PDAC samples vs. normal and CP samples. 2) Analysis of COL11A1 by immunohistochemistry using highly specific anti-proCOL11A1 antibodies. Findings: anti-proCOL11A1 stains stromal cells/cancer-associated fibroblasts (CAFs) of PDAC but it does not stain chronic benign condition (chronic pancreatitis) stromal cells, epithelial cells, or normal fibroblasts. 3) Evaluation of the discrimination ability of the antibody. Findings: anti-proCOL11A1 immunostaining accurately discriminates between PDAC and CP (AUC 0.936, 95% CI 0.851, 0.981). 4) Phenotypic characterization of proCOL11A1+ stromal cells co-staining with mesenchymal, epithelial and stellate cell markers on pancreatic tissue samples and cultured peritumoral pancreatic cancer stromal cells. Findings: ProCOL11A1+ cells present co-staining with mesenchymal, stellate and epithelial markers (EMT phenotype) in different proportions. Conclusions/Significance Detection of proCOL11A1 through immunostaining with this newly-developed antibody allows for a highly accurate distinction between PDAC and CP. Unlike other available antibodies commonly used to detect CAFs, anti-proCOL11A1 is negative in stromal cells of the normal pancreas and almost absent in benign inflammation. These results strongly suggest that proCOL11A1 is a specific marker for CAFs, and thus, anti-proCOL11A1 is a powerful new tool for cancer research and clinical diagnostics.

Performance of a Pneumolysin Enzyme-Linked Immunosorbent Assay for Diagnosis of Pneumococcal Infections

ABSTRACT A pneumolysin-specific enzyme-linked immunosorbent assay (PLY-ELISA) for the detection of pneumolysin in urine was developed and evaluated in comparison with the commercially available Binax Now Streptococcus pneumoniae test (Binax, Portland, ME) for the diagnosis of pneumococcal infections. Assay sensitivity was evaluated using urine from 108 patients with culture-confirmed pneumococcal infections. In adults, the sensitivity and specificity of the PLY-ELISA were 56.6% and 92.2%, respectively. In children with nasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 62.5% and 87.5%, respectively, while specificities were 94.4% and 27.8%, respectively. In children with nonnasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 68.7% and 93.7%, respectively, and test specificities were 94.1% and 41.2%, respectively. The persistence of pneumolysin in urine of pneumococcal pneumonia patients decreased significantly after 4 to 6 days of treatment. Our data suggest that combining the high specificity of the PLY-ELISA with the high sensitivity of the Binax Now S. pneumoniae test would enable pneumococcal infections to be accurately diagnosed in children.

The usefulness of new serum tumor markers in head and neck squamous cell carcinoma

Objective: To determine the usefulness of specific and reliable serum biomarkers to predict cervical lymph node metastasis. Methods: A cross-sectional study of cases and controls. Thirty-nine serum samples of head and neck squamous cell carcinoma were collected from patients during neoplasm resection. Another 10 serum samples were collected from healthy individuals as a control group. Selected serum biomarkers were E-cadherin, MMP-2, MMP-9, active MMP-13, and p53 autoantibodies. Results: We found a correlation between active MMP-13 (>685 pg/mL; ROC curve analysis 95% CI for sensitivity 79.6-99.3; specificity 49.2-95.1; positive predictive value 65-100; and negative predictive value 36-100) as well as the presence of p53 autoantibodies and lymph node metastasis. Multimarker analysis using MMP-13 and p53 autoantibodies together provided better sensitivity (76%) and specificity (100%). Conclusions: The combined determination of active MMP-13 and p53 autoantibodies could improve diagnosis of lymphatic metastasis in head and neck squamous cell carcinoma and aid therapeutic decision making.