A Unique Population of Regulatory T Cells in Heart Potentiates Cardiac Protection From Myocardial Infarction.

BACKGROUND: Regulatory T cells (Tregs), traditionally recognized as potent suppressors of immune response, are increasingly attracting attention because of a second major function: residing in parenchymal tissues and maintaining local homeostasis. However, the existence, unique phenotype, and function of so-called tissue Tregs in the heart remain unclear. METHODS: In mouse models of myocardial infarction (MI), myocardial ischemia/reperfusion injury, or cardiac cryoinjury, the dynamic accumulation of Tregs in the injured myocardium was monitored. The bulk RNA sequencing was performed to analyze the transcriptomic characteristics of Tregs from the injured myocardium after MI or ischemia/reperfusion injury. Photoconversion, parabiosis, single-cell T-cell receptor sequencing, and adoptive transfer were applied to determine the source of heart Tregs. The involvement of the interleukin-33/suppression of tumorigenicity 2 axis and Sparc (secreted acidic cysteine-rich glycoprotein), a molecule upregulated in heart Tregs, was further evaluated in functional assays. RESULTS: We showed that Tregs were highly enriched in the myocardium of MI, ischemia/reperfusion injury, and cryoinjury mice. Transcriptomic data revealed that Tregs isolated from the injured hearts had plenty of differentially expressed transcripts in comparison with their lymphoid counterparts, including heart-draining lymphoid nodes, with a phenotype of promoting infarct repair, indicating a unique characteristic. The heart Tregs were accumulated mainly because of recruitment from the circulating Treg pool, whereas local proliferation also contributed to their expansion. Moreover, a remarkable case of repeatedly detected T-cell receptor of heart Tregs, more than that of spleen Tregs, suggests a model of clonal expansion. Besides, HelioshighNrp-1high phenotype proved the mainly thymic origin of heart Tregs, with a small contribution of phenotypic conversion of conventional CD4+ T cells, proved by the analysis of T-cell receptor repertoires and conventional CD4+ T cells adoptive transfer experiments. The interleukin-33/suppression of tumorigenicity 2 axis was essential for sustaining heart Treg populations. Last, we demonstrated that Sparc, which was highly expressed by heart Tregs, acted as a critical factor to protect the heart against MI by increasing collagen content and boosting maturation in the infarct zone. CONCLUSIONS: We identified and characterized a phenotypically and functionally unique population of heart Tregs that may lay the foundation to harness Tregs for cardioprotection in MI and other cardiac diseases.

Characterization of Litopenaeus vannamei secreted protein acidic and rich in cysteine -like in WSSV infection.

Secreted protein acidic and rich in cysteine (SPARC) is an extracellular and non-structural glycoprotein. In shrimp, a significant function of SPARC in WSSV infection remains unclear. In this study, the full-length cDNA sequence of a secreted protein acidic and rich in cysteine -like was cloned from shrimp Litopenaeus vannamei (named as LvSPARC-L). LvSPARC-L contained an open reading frame of 1002 bp, encoding 333 amino acids. Bioinformatics analysis showed that LvSPARC-L contained a SPARC Ca2+-binding region in the C-terminus, a Kazal-type serine protease inhibitor domain and a BUD22 domain. Tissue distribution assay indicated that LvSPARC-L generally expressed in all tissues selected with a higher expression in hemocyte, stomach and pleoplod. In hepatopancreas and intestine, the relative expression of LvSPARC-L was significantly up-regulated following the WSSV challenge. Besides, the relative expression of viral immediately early gene IE1 and a late gene VP28 was significantly increased in the LvSPARCL-silenced shrimp. Furthermore, the relative expression of LvP53 and LvCaspase3 was extremely decreased in the stomach of dsLvSPARC-L treated shrimp, while that of LvP38 was not affected significantly. All data together suggest that LvSPARC-L might play an antiviral role by regulating apoptosis.

Bispecific Antibody Binding To RANKL and Osteonectin with Enhanced Localization to the Bone.

Therapeutics reducing bone turnover, such as denosumab (Dmab), an anti-RANKL antibody, can provide treatments for patients with bone destruction. However, some patients with osteoporosis or localized primary bone tumors and many patients with various types of bone-metastatic cancer display unsatisfactory responses to Dmab. For achieving greater efficiency of RANKL neutralization in the bone microenvironment by enhancing the distribution of Dmab to the bone, we reengineered Dmab by fusing with single-chain variable fragments of an antibody specific for osteonectin (On), which is abundantly expressed in osseous tissues. The bispecific antibody, Dmab-FvOn, showed a similar activity as Dmab in inhibiting RANKL as examined in an osteoclast differentiation assay. When administered to mice, Dmab-FvOn was found to localize in increased proportions at the endosteum of the bone where osteonectin is abundant. Our study suggests that by linking anti-RANKL with an osteonectin-targeting moiety, a greater proportion of the therapeutic effector can be distributed in the bone. Future studies are needed to investigate whether the bispecific antibody can achieve higher therapeutic efficacy and lower toxicity.

Secreted protein acidic and rich in cysteine facilitates age-related cardiac inflammation and macrophage M1 polarization.

To investigate the role of secreted protein acidic and rich in cysteine (SPARC) in age-related cardiac inflammation, we studied six groups of mice: young (3-5 mo old), middle-aged (10-12 mo old), and old (18-29 mo old) C57BL/6 wild-type (WT) and SPARC-null (Null) mice (n = 7-10/group). Cardiac function and structure were determined by echocardiography. The left ventricle was used for cytokine gene array and macrophage quantification by immunohistochemistry. Macrophage infiltration increased with age in WT (n = 5-6/group, P < 0.05 for young vs. old), but not in Null. Proinflammatory markers (Ccl5, Cx3cl1, Ccr2, and Cxcr3) increased in middle-aged and old WT, whereas they were increased only in old Null compared with respective young (n = 5-6/group, P < 0.05 for all). These results suggest that SPARC deletion delayed age-related cardiac inflammation. To further assess how SPARC affects inflammation, we stimulated peritoneal macrophages with SPARC (n = 4). SPARC treatment increased expression of proinflammatory macrophage M1 markers and decreased anti-inflammatory M2 markers. Echocardiography (n = 7-10/group) revealed an age-related increase in wall thickness of the left ventricle in WT (0.76 ± 0.02 mm in young vs. 0.91 ± 0.03 mm in old; P < 0.05) but not in Null (0.78 ± 0.01 mm in young vs. 0.84 ± 0.02 mm in old). In conclusion, SPARC deletion delayed age-related increases in macrophage infiltration and proinflammatory cytokine expression in vivo and in vitro. SPARC acts as an important mediator of age-related cardiac inflammation by increasing the expression of macrophage M1 markers and decreasing M2 markers.

The matricellular protein SPARC supports follicular dendritic cell networking toward Th17 responses.

Lymphnode swelling during immune responses is a transient, finely regulated tissue rearrangement, accomplished with the participation of the extracellular matrix. Here we show that murine and human reactive lymph nodes express SPARC in the germinal centres. Defective follicular dendritic cell networking in SPARC-deficient mice is accompanied by a severe delay in the arrangement of germinal centres and development of humoral autoimmunity, events that are linked to Th17 development. SPARC is required for the optimal and rapid differentiation of Th17 cells, accordingly we show delayed development of experimental autoimmune encephalomyelitis whose pathogenesis involves Th17. Not only host radioresistant cells, namely follicular dendritic cells, but also CD4(+) cells are the relevant sources of SPARC, in vivo. Th17 differentiation and germinal centre formation mutually depend on SPARC for a proper functional crosstalk. Indeed, Th17 cells can enter the germinal centres in SPARC-competent, but not SPARC-deficient, mice. In summary, SPARC optimizes the changes occurring in lymphoid extracellular matrix harboring complex interactions between follicular dendritic cells, B cells and Th17 cells.

Molecular cloning and functional analysis of an orange-spotted grouper (Epinephelus coioides) secreted protein acidic and rich in cysteine (SPARC) and characterization of its expression response to nodavirus.

Mammalian secreted protein acidic and rich in cysteine (SPARC) is the primary regulator of cell shape and cell adhesion to fibronectin. We, for the first time, report the complete sequencing of SPARC cDNA from orange-spotted grouper. Despite the difference in the lengths of the SPARC transcripts, all of the SPARC molecules encoded a signal peptide, follistain-like copper binding sequence (KGHK) domain, and extracellular domain. The grouper SPARC gene was differentially expressed in vivo and contributed differently to high-level expression of SPARC in muscle. Immunohistochemical staining demonstrated a decreased level of SPARC in nodavirus-infected grouper compared with healthy grouper. Comparative real-time polymerase chain reaction analyses of eye tissues of viral nervous necrosis grouper and healthy grouper were performed. Recombinant SPARC produced changes in grouper cell shape 24 h after treatment. The results provide new insight into the pathogenesis of nodavirus, and demonstrate an experimental rationale for SPARC characterization in nodavirus-infected grouper.

Identification of SPARC as a candidate target antigen for immunotherapy of various cancers.

To establish efficient anticancer immunotherary, it is important to identify tumor-associated antigens (TAAs) directing the immune system to attack cancer. A genome-wide cDNA microarray analysis identified that secreted protein acidic and rich in cysteine (SPARC) gene is overexpressed in the gastric, pancreatic and colorectal cancer tissues but not in their noncancerous counterparts. This study attempted to identify HLA-A24 (A*2402)-restricted and SPARC-derived CTL epitopes. We previously identified H-2K(d)-restricted and SPARC-derived CTL epitope peptides in BALB/c mice, of which H-2K(d)-binding peptide motif is comparable with that of HLA-A24 binding peptides. By using these peptides, we tried to induce HLA-A24 (A*2402)-restricted and SPARC-reactive human CTLs and demonstrated an antitumor immune response. The SPARC-A24-1(143-151) (DYIGPCKYI) and SPARC-A24-4(225-234) (MYIFPVHWQF) peptides-reactive CTLs were successfully induced from peripheral blood mononuclear cells by in vitro stimulation with these two peptides in HLA-A24 (A*2402) positive healthy donors and cancer patients, and these CTLs exhibited cytotoxicity specific to cancer cells expressing both SPARC and HLA-A24 (A*2402). Furthermore, the adoptive transfer of the SPARC-specific CTLs could inhibit the tumor growth in nonobese diabetic/severe combined immunodeficient mice bearing human cancer cells expressing both HLA-A24 (A*2402) and SPARC. These findings suggest that SPARC is a potentially useful target candidate for cancer immunotherapy.

Identification of the H2-Kd-restricted cytotoxic T lymphocyte epitopes of a tumor-associated antigen, SPARC, which can stimulate antitumor immunity without causing autoimmune disease in mice.

We previously reported that the secreted protein acidic and rich in cystein (SPARC) was overexpressed in melanoma in humans, and the serum SPARC level was useful as a novel tumor marker for melanoma. SPARC was also reported to be overexpressed in various human cancers. In this study, we asked whether SPARC-specific cytotoxic T lymphocytes (CTL) could induce antitumor immunity to SPARC-expressing tumor in mice or not as a preclinical study of SPARC-directed anticancer immunotherapy. Because of similarities in the structural motifs of major histocompatibility complex-binding peptides between H2-Kd and HLA-A24 (A*2402), the most common human leukocyte antigen class I allele in the Japanese population, we attempted to identify the H2-Kd-restricted SPARC epitope for CTL in BALB/c mice and we found that the mouse SPARC143-151 (DYIGPCKYI) and SPARC225-234 (MYIFPVHWQF) peptides could induce peptide-reactive CTL in BALB/c mice without causing autoimmune diseases. The immunization of mice with SPARC225-234 peptide-pulsed bone marrow-derived dendritic cells (BMDC) inhibited the growth of s.c. inoculated mouse mammary cancer cell line, N2C, expressing SPARC and these mice lived longer than the mice immunized with peptide-unpulsed BMDC. In conclusion, our study indicated that SPARC peptide-based cancer immunotherapy was effective and safe at least in a mouse tumor prevention model.

SPARC Is a New Myeloid-Derived Suppressor Cell Marker Licensing Suppressive Activities.

Myeloid-derived suppressor cells (MDSC) are well-known key negative regulators of the immune response during tumor growth, however scattered is the knowledge of their capacity to influence and adapt to the different tumor microenvironments and of the markers that identify those capacities. Here we show that the secreted protein acidic and rich in cysteine (SPARC) identifies in both human and mouse MDSC with immune suppressive capacity and pro-tumoral activities including the induction of epithelial-to-mesenchymal transition (EMT) and angiogenesis. In mice the genetic deletion of SPARC reduced MDSC immune suppression and reverted EMT. Sparc-/- MDSC were less suppressive overall and the granulocytic fraction was more prone to extrude neutrophil extracellular traps (NET). Surprisingly, arginase-I and NOS2, whose expression can be controlled by STAT3, were not down-regulated in Sparc-/- MDSC, although less suppressive than wild type (WT) counterpart. Flow cytometry analysis showed equal phosphorylation of STAT3 but reduced ROS production that was associated with reduced nuclear translocation of the NF-kB p50 subunit in Sparc-/- than WT MDSC. The limited p50 in nuclei reduce the formation of the immunosuppressive p50:p50 homodimers in favor of the p65:p50 inflammatory heterodimers. Supporting this hypothesis, the production of TNF by Sparc-/- MDSC was significantly higher than by WT MDSC. Although associated with tumor-induced chronic inflammation, TNF, if produced at high doses, becomes a key factor in mediating tumor rejection. Therefore, it is foreseeable that an unbalance in TNF production could skew MDSC toward an inflammatory, anti-tumor phenotype. Notably, TNF is also required for inflammation-driven NETosis. The high level of TNF in Sparc-/- MDSC might explain their increased spontaneous NET formation as that we detected both in vitro and in vivo, in association with signs of endothelial damage. We propose SPARC as a new potential marker of MDSC, in both human and mouse, with the additional feature of controlling MDSC suppressive activity while preventing an excessive inflammatory state through the control of NF-kB signaling pathway.

Molecular cloning of SC1: a putative brain extracellular matrix glycoprotein showing partial similarity to osteonectin/BM40/SPARC.

We describe the cloning of SC1, a novel cDNA that was selected from a rat brain expression library using a mixed polyclonal antibody directed against synaptic junction glycoproteins. SC1 detects a 3.2 kb mRNA expressed throughout postnatal development of the brain and present at high levels in the adult. In situ hybridization reveals that the SC1 mRNA is expressed widely in the brain and is present in many types of neurons. DNA sequence data suggest that the SC1 product is a secreted, calcium binding glycoprotein. Strikingly, the carboxy-terminal region of the SC1 protein shows substantial similarity to the extracellular matrix glycoprotein osteonectin/BM40/SPARC. These data are consistent with the hypothesis that SC1 is an extracellular matrix glycoprotein in the brain.

Micro-anatomical structure of the first spine of the dorsal fin of Atlantic bluefin tuna, Thunnus thynnus (Osteichthyes: Scombridae).

The first spine of the first dorsal fin (FS) of the Atlantic bluefin tuna (ABFT), Thunnus thynnus, is customarily used in age determination research because its transverse sections display well-defined growth marks. In this paper the FS structure was studied to explain its known dramatic age- and season-related morphological modifications, which are evidently caused by bone remodeling. Cross sections of samples from six adult ABFT were in part decalcified to be stained with histological, histochemical and immunohistochemical methods, and in part embedded in methyl-methacrylate to be either observed under a linear polarized light or microradiographed. FS showed an external compact bone zone and an inner trabecular bone zone. The compact bone zone consisted of an outer non-osteonic primary bone layer (C1) and an inner osteonic bone layer (C2). C1 was in turn characterized by alternate translucent and opaque bands. Evidence of spine bone remodeling was shown by the presence of osteoclasts and osteoblasts as well as by tartrate-resistant acid phosphatase (TRAP) positive bands at the boundary between old and newly formed bone. The examination of plain, i.e. not-fixed and not-decalcified, FS from 28 ABFT showed that the average thickness of C1 remained fairly constant during fish growth, whereas C2 increased significantly, indicating that the periosteal primary bone apposition is counterbalanced by the parallel bone remodeling occurring inside the compact bone zone. The present study revealed the structure of the ABFT FS and the pattern of its bone remodeling. Both of them underlay phenomena, never examined in detail before, such as the appearance followed by the progressive disappearance of growth bands.

Differential expression of osteonectin/SPARC during human prostate cancer progression.

The precise mechanism(s) involved in invasion and metastasis of prostate cancer (CaP) is poorly understood. Osteonectin [ON (also known as SPARC or BM-40)] is an antiadhesive protein known to be involved in cell-matrix interactions, migration, and angiogenesis. In this report, we studied the expression of ON in human prostate cell lines, primary tumors, and metastatic foci of CaP. Reverse transcription-PCR and nonradioactive in situ hybridization (ISH) techniques were used to determine ON gene expression. Immunohistochemistry was carried out using the polyclonal antibody LF37 and/or the monoclonal antibody ON-mAb. Low to moderate levels of ON mRNA and protein were observed in glandular epithelial cells of normal tissue as well as a few primary CaPs. However, high levels of ON mRNA and protein were observed in most of the CaP metastatic foci, both osseous and nonosseous. This correlated well with our findings that multiple different CaP cell lines including four CaP cell lines derived from metastases show high levels of ON gene expression. Furthermore, ISH analyses and cell-specific reverse transcription-PCR evaluation showed that both the luminal and basal cells express the ON gene. We conclude that the differential pattern of ON expression suggests that it may play an important role in the progression of CaP.

Osteonectin is an alpha-granule component involved with thrombospondin in platelet aggregation.

We previously showed that thrombospondin, a major alpha-granule glycoprotein of human platelets, forms a specific complex with osteonectin, a phosphoglycoprotein originally described in bone that is also present in human platelets. The storage organelles and the function of osteonectin in platelets are still unknown. In this study, using electron microscopy in combination with immunogold staining, the major storage organelle for platelet-secreted proteins, the alpha-granules. Furthermore, osteonectin was qualitatively and quantitatively assessed by studying normal platelets and the platelets from a patient with gray platelet syndrome. Gray platelet syndrome is a rare congenital bleeding disorder characterized by a selective deficiency in morphologically recognizable platelet alpha-granules and in the alpha-granule secretory proteins. Binding of an iodinated antiosteonectin monoclonal antibody to gray platelet proteins transferred to nitrocellulose from SDS-polyacrylamide gels showed no band corresponding to osteonectin compared to control platelets. Using a polyclonal antiosteonectin antibody-based radioimmunoassay, gray platelets contained 0.2 +/- 0.03 ng osteonectin per 10(6) platelets, which is only 20% of the normal platelet content of osteonectin (0.93 +/- 0.16 ng per 10(6) platelets). Study of the localization of osteonectin to the surface of human platelets demonstrated that a radioiodinated antiosteonectin polyclonal antibody bound specifically to thrombin-stimulated platelets but not to resting platelets. Binding was concentration-dependent, saturable (1710 +/- 453 binding sites per platelet, Kd = 1 microM), and inhibited by an excess of cold antiosteonectin polyclonal antibody. No binding was observed on the surface of thrombin-stimulated gray platelets. To gain further insights into the role of osteonectin released from activated platelets, the effect of an antiosteonectin polyclonal antibody was tested on the aggregation of washed platelets. F(ab')2 fragments from the antiosteonectin polyclonal antibody inhibited in a dose-dependent manner the aggregation of collagen-stimulated, washed human platelets without affecting collagen-induced platelet serotonin release. To characterize the mechanism through which antiosteonectin F(ab')2 fragments inhibit platelet aggregation, the expression of endogenous thrombospondin (TSP) on the surface of thrombin-activated platelets was studied using 125I-labeled anti-TSP monoclonal antibody P10. The endogenous surface expression of TSP to thrombin-stimulated platelets was significantly inhibited in the presence of antiosteonectin F(ab')2 fragments (6286 +/- 2065 molecules of P10 per platelet) compared to 11,230 +/- 766 molecules of P10 per platelet in the presence of nonimmune F(ab')2 fragments.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunochemical and tissue analysis of protease generated neoepitopes of BM-40 (osteonectin, SPARC) which are correlated to a higher affinity binding to collagens.

Proteolytic cleavage at single sites in the extracellular calcium-binding module of BM-40/SPARC/osteonectin either by an unknown endogenous protease (L197-L198) or several matrix metalloproteinases (E196-L197) was previously shown to enhance collagen binding activity 10-fold. Polyclonal rabbit antibodies were now obtained against synthetic peptide antigens containing either an N-terminal L197 or L198 and characterized by radioimmunoassay, ELISA, immunoblots and immunohistology. These neoepitope-specific antibodies reacted with proteolytically processed but not with uncleaved mouse and human BM-40. The cross-reaction between the two different neoepitopes was < 1%, indicating the immunodominant role of the N-terminal residues. Analysis of a basement membrane producing mouse tumor demonstrated extensive cleavage at the L198 site, which correlated with a calcium-dependent binding to the matrix. A variable degree of this cleavage was also detected in BM-40 obtained from adult mouse bone and several other tissues. Negligible or much lower levels of conversion were detected at the MMP-specific L197 site, however. Immunogold staining of mouse heart and a basement membrane-producing mouse tumor showed a distinct extracellular labeling for BM-40 and the L198 neoepitope but only a very weak reaction for the L197 neoepitope. This strongly indicates that these neoepitopes are generated in vivo and emphasizes a specific biological role for the proteolytic activation of BM-40.

Immunological screening of SPARC/Osteonectin in nonmineralized tissues.

SPARC/Osteonectin is a major bone-related protein that is also present in nonmineralized tissues and in platelets. As compared to bone SPARC/Osteonectin, SPARC/Osteonectin from platelets presents a slightly lower electrophoretic mobility in SDS-PAGE and a 100-fold decreased affinity for a unique monoclonal antibody, Mab2 (Malaval et al. 1991). To check the tissular diversity of SPARC/Osteonectin, protein extracts from bovine bone, nonmineralized tissues, and platelets were screened by immunoblotting and immunoradiometric assay, with Mab2 and three other monoclonal antibodies recognizing distinct epitopes. The SPARC/Osteonectin secreted by a human osteosarcoma cell line (MG63) was also tested. In all the nonmineralized tissues tested (gut, bone marrow, tendon, mesentery, artera, lens, skin, liver, and cornea), SPARC/Osteonectin presents the same immunoreactivity and electrophoretic mobility as in bone. The heavier molecular weight and Mab2-negative form present in platelets seems to be unique to this cell type. Osteosarcoma cell extracts and conditioned media give the same results as bone extracts, indicating that the low molecular weight and Mab2-positive form of SPARC/Osteonectin present in most tissues does not result from proteolytic cleavage in the matrix, but is secreted as such. Bone and platelet SPARC/Osteonectin present different patterns of sensitivity to glycosidases, suggestive of a difference in N-glycosylation. However, these treatments do not affect the decreased affinity of Mab2 for platelet SPARC/Osteonectin, which is not likely to be related to difference in N-glycosylation.

Distribution of SPARC in normal and neoplastic human tissue.

SPARC (Secreted Protein, Acidic and Rich in Cysteine)/osteonectin is a secreted glycoprotein that exhibits restricted expression in murine adult and embryonic tissues and is associated with cell migration, matrix mineralization, steroid hormone production, cell cycle regulation, and angiogenesis. We produced a monoclonal antibody, MAb SSP2, against a Ca(2+)-binding region of SPARC and evaluated the immunoreactivity of normal and malignant tissue from 118 human samples. In normal tissue we found restricted and moderate reactivity with SSP2 in steroidogenic cells, chondrocytes, placental trophoblasts, vascular smooth muscle cells, and endothelial cells. Strong reactivity was found in fibrocytes and endothelial cells involved in tissue repair and in invasive malignant tumors, including those of the gastrointestinal tract, breast, lung, kidney, adrenal cortex, ovary, and brain. We conclude that SSP2 is a useful reagent for detection of SPARC in human tissue. Given the broad reactivity of malignant tissues, we propose that SPARC expression might contribute to some aspects of tumor progression.

Functional analysis of the matricellular protein SPARC with novel monoclonal antibodies.

SPARC (osteonectin, BM-40) is a matricellular glycoprotein that is expressed in many embryogenic and adult tissues undergoing remodeling or repair. SPARC modulates cellular interaction with the extracellular matrix (ECM), inhibits cell adhesion and proliferation, and regulates growth factor activity. To explore further the function and activity of this protein in tissue homeostasis, we have developed several monoclonal antibodies (MAbs) that recognize distinct epitopes on SPARC. The MAbs bind to SPARC with high affinity and identify SPARC by ELISA, Western blotting, immunoprecipitation, immunocytochemistry, and/or immunohistochemistry. The MAbs were also characterized in functional assays for potential alteration of SPARC activity. SPARC binds to collagen I and laminin-1 through an epitope defined by MAb 293; this epitope is not involved in the binding of SPARC to collagen III. The other MAbs did not interfere with the binding of SPARC to collagen I or III or laminin-1. Inhibition of the anti-adhesive effect of SPARC on endothelial cells by MAb 236 was also observed. Functional analysis of SPARC in the presence of these novel MAbs now confirms that the activities ascribed to this matricellular protein can be assigned to discrete subdomains.

Osteocalcin and osteonectin immunoreactivity in the diagnosis of osteosarcoma.

Osteosarcomas (OSAs) can be difficult to distinguish histologically from tumors with significantly different biologic potentials and treatment protocols. The correct diagnosis of OSA relies on identification of malignant osteoblasts that are capable of producing neoplastic bone. To determine the use of immunohistochemistry for the diagnosis of OSA, 106 tumors from the Massachusetts General Hospital and the University of Vermont were immunostained with monoclonal antiosteocalcin (OC) and antiosteonectin (ON) antibodies. They included 42 OSAs, 25 non-bone-forming sarcomas, 24 other malignant tumors including lymphomas, carcinomas, and melanomas, and 15 benign bone tumors. Cytoplasmic staining with OC showed 70% sensitivity and 100% specificity, while staining with ON showed 90% sensitivity and 54% specificity for bone-forming tumors, consistently staining cell types other than osteoblasts. Of the OSAs, 83% demonstrated matrix staining with one or both antibodies, whereas dense collagen was negative for both antibodies in all tumors. We conclude that tumor cell cytoplasmic staining with monoclonal OC may be helpful in distinguishing OSAs from other malignancies, and staining of extracellular matrix for OC and ON antibodies concurrently may help distinguish bone matrix from dense collagen.

Cell differentiation and bone protein synthesis in the lungs of sheep with spontaneous calcinosis.

The lungs of three sheep with spontaneous enzootic calcinosis induced by the calcinogenic plant Nierembergia veitchii (Nv) were examined electron microscopically and immunohistochemically. The main ultrastructural changes were activation of fibroblasts and modified smooth muscle cells (SMCs) in the pulmonary interstitium, with an increase in extracellular matrix and precipitation of calcium, either in a laminated pattern or as amorphous aggregates. Macrophages and multinucleated giant cells, some with calcium crystals in the cytoplasm, were found in areas of increased extracellular matrix. Thickening and replication of the basal lamina of capillaries were prominent. The bone proteins osteocalcin, osteopontin and osteonectin were detected immunohistochemically in the cytoplasm of activated fibroblasts, in modified SMCs and in the extracellular matrix. It is suggested that 1,25-dihydroxyvitamin D(3)in Nv induces cellular differentiation and the synthesis of a calcifiable matrix.

Defective stromal remodeling and neutrophil extracellular traps in lymphoid tissues favor the transition from autoimmunity to lymphoma.

Altered expression of matricellular proteins can become pathogenic in the presence of persistent perturbations in tissue homeostasis. Here, we show that autoimmunity associated with Fas mutation was exacerbated and transitioned to lymphomagenesis in the absence of SPARC (secreted protein acidic rich in cysteine). The absence of SPARC resulted in defective collagen assembly, with uneven compartmentalization of lymphoid and myeloid populations within secondary lymphoid organs (SLO), and faulty delivery of inhibitory signals from the extracellular matrix. These conditions promoted aberrant interactions between neutrophil extracellular traps and CD5(+) B cells, which underwent malignant transformation due to defective apoptosis under the pressure of neutrophil-derived trophic factors and NF-κB activation. Furthermore, this model of defective stromal remodeling during lymphomagenesis correlates with human lymphomas arising in a SPARC-defective environment, which is prototypical of CD5(+) B-cell chronic lymphocytic leukemia (CLL).