Proton pump inhibitors protect mice from acute systemic inflammation and induce long-term cross-tolerance.

Incidence of sepsis is increasing, representing a tremendous burden for health-care systems. Death in acute sepsis is attributed to hyperinflammatory responses, but the underlying mechanisms are still unclear. We report here that proton pump inhibitors (PPIs), which block gastric acid secretion, selectively inhibited tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) secretion by Toll-like receptor (TLR)-activated human monocytes in vitro, in the absence of toxic effects. Remarkably, the oversecretion of IL-1ß that represents a hallmark of monocytes from patients affected by cryopyrin-associated periodic syndrome is also blocked. Based on these propaedeutic experiments, we tested the effects of high doses of PPIs in vivo in the mouse model of endotoxic shock. Our data show that a single administration of PPI protected mice from death (60% survival versus 5% of untreated mice) and decreased TNF-α and IL-1ß systemic production. PPIs were efficacious even when administered after lipopolysaccharide (LPS) injection. PPI-treated mice that survived developed a long-term cross-tolerance, becoming resistant to LPS- and zymosan-induced sepsis. In vitro, their macrophages displayed impaired TNF-α and IL-1ß to different TLR ligands. PPIs also prevented sodium thioglycollate-induced peritoneal inflammation, indicating their efficacy also in a non-infectious setting independent of TLR stimulation. Lack of toxicity and therapeutic effectiveness make PPIs promising new drugs against sepsis and other severe inflammatory conditions.

Lack of specificity of endoglin expression for tumor blood vessels.

A monoclonal antibody (MAb; A11) has been raised following mouse immunization with cultured human microvascular endothelial cells. The MAb showed a strong positivity within tumor vessels in glioblastoma and breast carcinoma samples, and the distribution was consistent with antigen association with vascular endothelial cells. A purification procedure of the antigen was developed starting from DG-RSV-LT-2, an immortalized human endothelial cell line. Molecular mass, N-terminal sequence of the purified antigen and localization on endothelial cell surface allowed identification with human endoglin (CD105). Flow cytometry analysis of a group of normal and transformed cell lines showed that, besides endothelial cells and myelocytic leukemia cells already shown to be positive, fetal fibroblasts, choriocarcinoma, fibrosarcoma and rhabdomyosarcoma cell lines were also positive for this antigen. Immunohistochemic analysis of several normal adult tissues revealed a more extensive presence of the antigen in normal vessels compared to that described with previously characterized antibodies. In fact, even though the staining was weaker than in tumor tissues, all tissues were found to be positive, at least in microvessels, except for normal breast. Moreover, in some tissues (glands and reproductive tract) a positive reaction was observed in the stroma. Since endoglin has been proposed as a possible target for antiangiogenic therapy in tumor patients and our data demonstrate a sizable amount of endoglin in normal vessels and stroma, its clinical use should be carefully reevaluated.

A high-affinity human antibody that targets tumoral blood vessels.

Angiogenesis is a characteristic feature of many aggressive tumors and of other relevant disorders. Molecules capable of specifically binding to new-forming blood vessels, but not to mature vessels, could be used as selective vehicles and would, therefore, open diagnostic and therapeutic opportunities. We have studied the distribution of the ED-B oncofetal domain of fibronectin, a marker of angiogenesis, in four different tumor animal models: the F9 murine teratocarcinoma, SKMEL-28 human melanoma, N592 human small cell lung carcinoma, and C51 human colon carcinoma. In all of these experimental models we observed accumulation of the fibronectin isoform containing the ED-B domain around neovascular structures when the tumors were in the exponentially growing phase, but not in the slow-growing phase. Then we performed biodistribution studies in mice bearing a subcutaneously implanted F9 murine teratocarcinoma, using a high-affinity human antibody fragment (L19) directed against the ED-B domain of fibronectin. Radiolabeled L19, but not an irrelevant anti-lysozyme antibody fragment (D1.3), efficiently localizes in the tumoral vessels. The maximal dose of L19 accumulated in the tumor was observed 3 hours after injection (8.2% injected dose per gram). By virtue of the rapid clearance of the antibody fragment from the circulation, tumor-to-blood ratios of 1.9, 3.7, and 11.8 were obtained at 3, 5, and 24 hours, respectively. The tumor-targeting performance of L19 was not dose-dependent in the 0.7 to 10 microg range of injected antibody. The integral of the radioactivity localized in tumoral vessels over 24 hours was greater than 70-fold higher than the integral of the radioactivity in blood over the same time period, normalized per gram of tissue or fluid. These findings quantitatively show that new-forming blood vessels can selectively be targeted in vivo using specific antibodies, and suggest that L19 may be of clinical utility for the immunoscintigraphic detection of angiogenesis in patients.

Tumor targeting potential of the monoclonal antibody BC-1 against oncofetal fibronectin in nude mice bearing human tumor implants.

BACKGROUND: The immunoglobulin G1 (IgG1) monoclonal antibody (MoAb) BC-1 detects human oncofetal fibronectin, which has extremely restricted distribution in normal adult tissues and is highly expressed in fetal and tumor tissues. METHODS: We studied the biodistribution of 125I-labeled MoAb BC-1 in nude mice bearing subcutaneous human tumor implants of U87MG high-grade astrocytoma and SKMel28 melanoma. 125I-BC-1 was injected either intraperitoneally (i.p.) or intravenously (i.v.), and biodistribution was measured up to 144 hours after injection. In animals bearing SKMel28 implants, tumor targeting was also evaluated by in vivo imaging of the whole mouse by using a dedicated device based on transmitted light excitation after i.v. injection of MoAb BC-1 conjugated with the infrared fluorophore, CY7-bis(N-hydroxy-succinimido)-ester. RESULTS: 125I-BC-1 showed favorable uptake in the human tumor implants, reaching a maximum of 5.27 +/- 0.48% ID/g in the U87MG astrocytoma (72 hours after i.p. injection). The highest uptake in the SKMel28 melanoma implants was 3.49 +/- 0.25% ID/g (24 hours after i.v. injection). Microautoradiography of tumor specimens obtained after administration of 125I-BC-1 clearly showed radioactivity uptake within the two tumors replicating the same pattern of distribution as that of the oncofetal fibronectin shown by immunohistochemistry with MoAb BC-1. Nonspecific uptake of 125I-BC-1 in the bone marrow and skeletal muscle was much lower than in the tumors. In vivo imaging with the fluorophore-labeled MoAb clearly visualized the tumor implants 72-120 hours after i.v. injection. CONCLUSIONS: The experimental results obtained in this study demonstrate the favorable tumor targeting potential in vivo of the radiolabeled MoAb BC-1, a useful marker of neo angiogenesis induced by cancer.

Targeting by affinity-matured recombinant antibody fragments of an angiogenesis associated fibronectin isoform.

The oncofetal fibronectin (B-FN) isoform is present in vessels of neoplastic tissues during angiogenesis but not in mature vessels. B-FN could therefore provide a target for diagnostic imaging and therapy of cancer. Phage display libraries have been used to isolate human antibody fragments with pan-species recognition of this isoform. We describe the use of these fragments in nude mice to target an aggressive tumor (grafted F9 murine teratocarcinoma). Imaging in real time was done by infrared photodetection of a chemically coupled fluorophore. The targeting was improved by use of affinity-matured fragments with low kinetic dissociation rates (koff = 1.5 x 10(-4) s-1) and also by engineering dimeric fragments via a C-terminal amphipathic helix.

The alternative splicing pattern of the tenascin-C pre-mRNA is controlled by the extracellular pH.

Alternative splicing of primary transcripts is an ubiquitous and reversible mechanism for the generation of multiple protein isoforms from single genes. Here we report that in cultured normal human fibroblasts, small pH variations of the culture medium (from 7.2 to 6.9) strikingly modify the alternative splicing pattern of the tenascin-C primary transcript. Since such extracellular pH variations occur in many normal and pathological conditions, microenvironmental pH may be an important element for the regulation of RNA alternative splicing in vivo.

Human tenascin gene. Structure of the 5'-region, identification, and characterization of the transcription regulatory sequences.

This report describes the genomic organization of the 5'-region of the human tenascin-C (TN) gene and the functional characterization of its promoter. Approximately 2300 base pairs of the TN gene 5'-flanking region have been cloned and sequenced. This genomic region contains several potential binding sites for transcription factors. By primer extension and S1 nuclease analysis we have localized the transcription start site. The first exon of the TN gene (179 base pairs long) is present in the two major TN transcripts, showing that the expression of these two mRNAs is regulated by a single promoter. The 220 bases upstream to the transcription start site are equally active in directing the expression of chloramphenicol acetyltransferase (CAT) reporter gene in TN producer and nonproducer cells. Using deletion fragments of the human 5'-flanking region we have shown the presence of putative "silencer" elements in the -220 to -2300 region active in both TN producer and nonproducer cell lines. Furthermore, we have demonstrated that the selective transcription in TN producing cells requires the presence of a 1.3-kilobase portion of the TN gene intron 1 in the CAT expression vectors. These findings indicate that complex mechanisms control the transcriptional regulation of TN gene.

Cell-cycle dependent alternative splicing of the tenascin primary transcript.

Functionally different tenascin (TN) isoforms may be generated by alternative splicing of the TN primary transcript. In fact, it has been demonstrated that only the larger TN isoform containing the alternatively spliced region induces loss of focal adhesion in cultured cells and seems able to facilitate cell migration. Recent studies have shown that the higher molecular mass TN isoform is a marker of stromal cell proliferation in hyperplastic and neoplastic breast tissues. This finding prompted us to study the pattern of TN alternative splicing in proliferating and non-proliferating cultured fibroblasts. Here, we show that the mitogenic stimulation of fibroblasts with serum or cytokines leads to an early and striking modification in the steady-state levels of the two major TN mRNAs. We also show that de novo protein synthesis is not necessary for this modification, indicating that it is a "primary response" event. Similarly, mitogenic stimulation induces changes both in synthesis and accumulation of the different TN isoforms.

Production and characterization of monoclonal antibodies specific for different epitopes of human tenascin.

We have obtained and characterized 11 monoclonal antibodies (mAbs) specific for different domains of human tenascin (TN). Five of these mAbs reacted with epitopes contained in the TN area that undergoes alternative splicing and are thus able to recognize specific TN isoforms. These mAbs are a useful tool to study the expression and distribution of TN and its different isoforms in normal and pathological tissues.

Human amnion epithelial cells assemble tenascins and three fibronectin isoforms in the extracellular matrix.

Monoclonal antibodies (MAb) were used to show that cultured human amnion epithelial (HuA) cells produce tenascins (Tn) and isoforms of cellular fibronectin (cFn). Tn polypeptides of M(r) 280,000 and 190,000, assembled into extracellular matrix (ECM) but not secreted into the culture medium by HuA cells, were electrophoretically similar to those produced by human fibroblasts as revealed with domain-specific MAbs. The results suggested that most Fn produced by HuA cells contained the extradomain (ED) A and an oncofetal domain but only a minor fraction EDB. In immunofluorescence Tn and Fn were seen in different cytoplasmic granules upon monensin-induced intracellular accumulation. Tn appeared to be deposited in the ECM in colocalization with Fn but distinctly slower. The present results show that cultured normal human epithelial cells synthesize Tn and three isoforms of cFn and secrete them by using different cytoplasmic pathways.

Tenascin and fibronectin distribution in human normal and pathological synovium.

Tenascin is a glycoprotein found mainly in the extracellular matrix of developing and malignant tissues. The distribution of this molecule in normal and pathological synovia from patients with osteoarthritis (OA) and rheumatoid arthritis (RA) was investigated by indirect immunofluorescence utilizing specific monoclonal antibodies. The same technique was used to study total fibronectin (tFn) in synovial tissues as well as ED-A and ED-B containing fibronectin (Fn) isoforms (A-Fn, B-Fn), generated by alternative splicing of pre-mRNA. Tenascin was found in normal synovium just beneath the whole lining cell layer. However, a higher density and spreading pattern of distribution was observed in OA and RA sections. A-Fn and B-Fn isoforms were prominent and widespread throughout the normal synovial lining; in hypercellular synovial lining (in RA and OA samples), A-Fn and B-Fn were also observed spreading in the sublining, as well as tFn.

A simple procedure for tenascin purification.

Here we describe a two-step procedure for purification of human tenascin from conditioned medium of the SK-MEL-28 human melanoma cell line. The first step consists in passing the conditioned media through two chromatography columns connected in sequence. The first is a large capacity gelatin--Sepharose affinity chromatography column (to remove fibronectin), the second, over which the unbound material from the first column flows directly, is a hydroxyapatite chromatography column. Under these conditions, all tenascin present in the conditioned medium binds to the hydroxyapatite chromatography column from which it is then eluted by a 5-300 mM sodium phosphate gradient. With this step, we obtain a crude tenascin preparation, concentrated about 20 times with respect to the starting conditioned medium, and in which tenascin represents more than 50% of the total protein. The second step consists of two sequential precipitations with 6% and 12.8% poly(ethylene glycol). After this step, tenascin is more than 95% pure and does not show any contamination of chondroitin-sulfate-containing proteoglycans that are known to bind to it. From 21 medium we obtain about 3-4 mg tenascin which corresponds to a yield of about 40-50%. This procedure gives a higher yield, is simpler with respect to procedures previously described, avoids the exposure of the protein to denaturing agents or harsh conditions and could be used for purification of tenascin from the conditioned media of other cell lines. Thus, this procedure may represent a simple and useful tool for the preparation of tenascin to study its biological functions.

Differential expression of the fibronectin isoform containing the ED-B oncofetal domain in normal human fibroblast cell lines originating from different tissues.

Fibronectin (FN) polymorphism is due both to alternative splicing of three sequences (ED-A, ED-B, and IIICS) of the primary transcript and to post-translational modifications. The FN isoform containing the ED-B sequence (B-FN), while having an extremely restricted distribution in normal adult tissues, has a high expression in fetal and tumor tissues. On a panel of non-fetal skin, fetal skin, and fetal lung fibroblast cell lines we have studied, through S1-nuclease protection analysis, the expression of the ED-B containing FN mRNA as well as the expression of the ED-B containing FN isoform through immunoblotting and immunofluorescence techniques, using domain specific monoclonal antibodies. The results show that the expression of B-FN in the different fibroblast cell lines has an extremely great variability depending on the developmental stage of the donor and on the tissue of origin. Moreover, we found that SV-40-transformed fibroblasts present a higher expression of B-FN mRNA with respect to their normal counterparts. An increase in the relative amount of the B-FN isoform in normal human fibroblasts was also obtained by treatment with transforming growth factor-beta.

Procedure for the purification of the fibronectin proteolytic fragments containing the ED-B oncofetal domain.

Fibronectin (FN) is the blend of structurally different molecules (isoforms) whose makeup varies depending on the FN sources. Fibronectin polymorphism is caused by three sequences (called ED-A, ED-B, and IIICS) which may be included or excluded from the FN molecule depending on the alternative splicing patterns of a single primary transcript. The sequence ED-B, which is a complete type III repeat of 91 amino acids, presents some interesting peculiarities: it is the most conserved FN region and, in normal adult tissues, the ED-B-containing FN has an extremely restricted distribution while having a much greater expression in fetal and tumor tissues (Carnemolla et al., 1989, J. Cell Biol. 108, 1139-1148), suggesting that the ED-B sequence may confer to the FN molecules specific biological activities required during ontogenesis and oncogenetic processes. Here we describe a detailed procedure to purify fibronectin fragments containing the ED-B sequence. These purified fragments are useful reagents in the study of the biological function(s) of the ED-B-containing FN molecules.

Requirement for two different cell-binding domains in fibronectin for neurite extension of neuronal derivative cells.

Some neuron-derived cells, such as neuroblastoma cells, adhere and extend neurites on fibronectin (FN) substrata by processes that can be independent of binding to the Arg-Gly-Asp-Ser sequence (RGDS in FN) and independent of proteoglycan/ganglioside-binding activities of FN. Proteolytic fragments of various FNs have been used in this study to map a new adhesion-promoting domain in FNs that may be neural cell-specific. A thermolysin-generated fragment of human plasma FN (F110 containing the RGDS domain) or the analagous fragment from transformed human cell FN (F120, also containing the alternately spliced extra domain b[EDb]) facilitate RGDS-independent adherence and neurite extension of human neuroblastoma cells and an F11 hybrid neuronal line (by fusion of mouse neuroblastoma cells with rat dorsal root ganglion neurons) as effectively as adherence and neurite extension on intact FN. Since neither F110 nor F120 contains sequences from the alternately spliced IIICS region of FN, neurite-promoting activity in these fragments cannot be ascribed to a recently discovered cell-binding domain in this region. Furthermore, F120 could be cleaved into two subfragments retaining virtually all the sequence of the parent fragment: F35 from the C terminus of F120 containing the RGDS domain, and F90 from the N terminus containing most of the EDb region bordering the thermolysin cleavage site. These neuronal cells could adhere but not extend neurites on substrata coated with either F35 or F90 alone while 3T3 cells could adhere only on F35. Mixtures of F35 and F90 on substrata could reconstitute some, but not nearly all, of the neurite-promoting activity of F120. Therefore, these data identify a new cell-binding domain in common sequences of FNs on the N-terminal side of EDb and demonstrate cooperativity between this RGDS-independent domain and the RGDS-dependent domain for maximal differentiation of these neuron-derived cells. Several possibilities for a receptor directed to this new domain are discussed.

A tumor-associated fibronectin isoform generated by alternative splicing of messenger RNA precursors.

Fibronectin (FN) represents the mixture of a number of structurally different molecules (isoforms) whose make-up varies depending on the FN sources. FN from cultured transformed human cells has a very different isoform composition with respect to its normal counterpart. In fact, SV-40-transformed WI-38VAI3 human fibroblasts produce high levels of a FN isoform (B-FN) which is very poorly expressed in their normal, WI-38, counterpart. We have recently demonstrated that the B-FN isoform derives from a differential splicing pattern of the FN primary transcript which leads, in transformed cells, to a high level expression of the exon ED-B (Zardi, L., B. Carnemolla, A. Siri, T. E. Petersen, G. Paolella, G. Sebastio, and F. E. Baralle. 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:2337-2342). Here we report on the production and characterization of a monoclonal antibody (BC-1) which recognizes an epitope within the protein sequence coded for by the ED-B exon. This monoclonal antibody makes it possible to carry out immunohistochemical analysis of the distribution of the ED-B-containing FN isoform (B-FN) in human tissues. The results show that while in normal, adult, human tissues total FN has a widespread distribution, the B-FN isoform is restricted only to synovial cells, to some vessels and areas of the interstitium of the ovary, and to the myometrium. On the contrary, the B-FN isoform has a much greater expression in fetal and tumor tissues. These results demonstrate that, in vivo, different FN isoforms have a differential distribution and indicate that the B-FN isoform may play a role in ontogenesis and oncogenetic processes.

Transforming growth factor beta regulates the levels of different fibronectin isoforms in normal human cultured fibroblasts.

Fibronectin (FN) polymorphism is caused by alternative splicing patterns in at least three regions of the primary transcript of a single gene. Using a monoclonal antibody (Mab) specific for an FN segment (ED-A), that can be included or omitted from the molecule depending on the pattern of splicing, we have examined whether transforming growth factor beta (TGF-beta) and dexamethasone, which are both known to increase the level of total FN, regulate the levels of different FN isoforms. We found that, while dexamethasone does not significantly change the ratio between the total FN and the ED-A containing FN, TGF-beta preferentially increases the expression of the FN isoform containing the ED-A sequence.

Large-scale procedure for the purification of fibronectin domains.

Human plasma fibronectin is composed of at least seven distinct domains, with affinities for different macromolecules and cell surfaces. Here we describe in detail a simple high-yield procedure for the purification of large amounts of fibronectin domains. This involves thermolysin digestion of the fibronectin molecule followed by the purification of the domain using mainly hydroxyapatite chromatography columns. This procedure represents a great simplification over those previously reported.

DNA-binding domains of human plasma fibronectin. pH and calcium ion modulation of fibronectin binding to DNA and heparin.

We have studied the binding of fibronectin and its thermolysin fragments to DNA and heparin. Elution of polypeptides bound to DNA-cellulose and heparin-Sepharose affinity chromatography columns was performed by NaCl linear gradients in buffers at different pH and in the presence and absence of calcium ions. The NaCl concentration required to elute fibronectin from both types of column increased as the pH decreased. Fibronectin was not retained on DNA-cellulose or heparin-Sepharose affinity chromatography columns using a buffer containing physiological concentrations of Ca2+, Mg2+ and NaCl, at pH 7.4. On the other hand at pH 6.4 in conditions of physiological ionic strength, fibronectin was retained by both columns, eluting from the DNA-cellulose at 280 mM NaCl and from the heparin-Sepharose column at 210 mM. Furthermore, we have studied the interaction of thermolysin-digested fibronectin both with DNA-cellulose and heparin-Sepharose using the above procedure. The results demonstrate that there are four distinct domains, which interact both with DNA and heparin. We also report here the modulation by pH and Ca2+ ions of the interaction with DNA and heparin of these different domains.

Elution of fibronectin proteolytic fragments from a hydroxyapatite chromatography column. A simple procedure for the purification of fibronectin domains.

Human plasma fibronectin is composed of at least five distinct domains which we refer to as Hep-1/Fib-1, Gel, Cell, Hep-2 and Fib-2 depending on their affinity for heparin (Hep), gelatin (Gel), the cell surface (Cell) or fibrin (Fib). These domains are aligned from the NH2 to the COOH terminus in the above order and can be separated from each other by mild proteolytic digestion. We have studied the elution of fibronectin thermolysin digest from a hydroxyapatite column using a linear gradient (0.5-190 mM) of sodium phosphate buffer. The five major fibronectin domains were eluted from the hydroxyapatite chromatography column in the following order: Gel, Fib-2, Cell, Hep-1/Fib-1 and Hep-2. They were identified on the basis of their molecular mass, affinity to different macromolecules and reaction with domain-specific monoclonal antibodies. All domains except the Cell and Hep-2 domains eluted as single homogeneous peaks. The Cell domain eluted as two different peaks and the Hep-2 domain eluted as four different peaks. This is the first time that heterogeneity of these two domains has been observed. Since chromatography of a fibronectin thermolysin digest on a hydroxyapatite column provides a good separation of the five major fibronectin domains, we have elaborated a procedure in which each fibronectin domain is purified by no more than two steps; hydroxyapatite and molecular exclusion chromatography. Fractionation of fibronectin proteolytic digest on a hydroxyapatite chromatography column should be of great value in the comparative analysis of fibronectin from different sources and in the study of fibronectin heterogeneity. Its use in combination with molecular exclusion chromatography offers a simple and high-yield method for the purification of large amounts of fibronectin domains.