Identification of synovium‐specific homing peptides by in vivo phage display selection

Objective

To identify homing peptides specific for human synovium that could be used as targeting devices for delivering therapeutic/diagnostic agents to human joints.

Methods

Human synovium and skin were transplanted into SCID mice. A disulfide‐constrained 7–amino acid peptide phage display library was injected intravenously into the animals and synovial homing phage recovered from synovial grafts. Following 3–4 cycles of enrichment, DNA sequencing of homing phage clones allowed the identification of specific peptides that were synthesized by a‐fluorenylmethyloxycarbonyl chemistry and used in competitive in vivo assays and immunohistochemistry analyses.

Results

We isolated synovial homing phages displaying specific peptides that distinctively bound to synovial but not skin or mouse microvascular endothelium (MVE). They retained their tissue homing specificity in vivo, independently from the phage component, the original pathology of the transplanted tissue, and the degree of human/murine graft vascularization. One such peptide (CKSTHDRLC) maintained synovial homing specificity both when presented by the phage and as a free synthetic peptide. The synthetic peptide also competed with and inhibited in vivo the binding of the parent phage to the cognate synovial MVE ligand.

Conclusion

This is the first report describing peptides with homing properties specific for human synovial MVE. This was demonstrated using a novel approach targeting human tissues, transplanted into SCID mice, directly by in vivo phage display selection. The identification of such peptides opens the possibility of using these sequences to construct joint‐specific drug delivery systems that may have considerable impact in the treatment of arthritic conditions.

     

Peptides targeting inflamed synovial vasculature attenuate autoimmune arthritis

Autoimmune diseases, such as rheumatoid arthritis, frequently target one major tissue/organ despite the systemic nature of the immune response. This is particularly perplexing in the case of ubiquitously distributed antigens invoked in arthritis induction. We reasoned that selective targeting of the synovial joints in autoimmune arthritis might be due in part to the unique attributes of the joint vasculature. We examined this proposition using the adjuvant-induced arthritis model of human rheumatoid arthritis, and profiled the synovial vasculature using ex vivo and in vivo screening of a defined phage peptide-display library. We identified phage that preferentially homed to the inflamed joints. The corresponding synthetic peptides showed binding to the joint-derived endothelial cells, as well as specificity in inhibiting binding of the respective phage to the synovial vasculature. Intriguingly, the treatment of arthritic rats with one such peptide resulted in efficient inhibition of the progression of arthritis. The suppression of arthritis was attributable in part to the peptide-induced reduction of T-cell trafficking into the joints and the inhibition of angiogenesis. This peptide differed in sequence, in receptor binding specificity, and in angiogenesis/inflammation-related cell signaling from the previously characterized arginine-glycine-aspartic acid–containing peptide. Thus, our study reveals joint-homing peptides that can be further exploited for the selective delivery of antiarthritic agents into the inflamed joints to enhance their efficacy while reducing systemic toxicity, and also for examining intricacies of the pathogenesis of arthritis. This approach can be customized for application to other organ-specific autoimmune diseases as well.     

Effects of tumor necrosis factor-alpha, interleukin 1beta, and activated peripheral blood mononuclear cells on the expression of adhesion molecules and recruitment of leukocytes in rheumatoid synovial xenografts in SCID mice.

OBJECTIVE: To examine expression of adhesion molecules within human synovial xenografts in vivo after injection of human cytokines or peripheral blood mononuclear cells (PBMC). METHODS: Rheumatoid synovium was transplanted into subcutaneous pouches in the ears of severe combined immunodeficient (SCID) mice. Tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), or PBMC were injected into the xenografts. The grafts were assessed by immunohistochemistry and quantitative video image analysis. RESULTS: TNF-alpha, IL-1beta, and concanavalin A activated PBMC increased the expression of vascular cellular adhesion molecule 1 and intercellular adhesion molecule 1 significantly on vessels and nonvascular synovial cells compared with injection of medium only. Vascular expression of E-selectin, but not of MHC Class II, was also increased. These stimuli also induced the migration of murine leukocytes into the xenografts. CONCLUSION: Rheumatoid synovial xenografts in SCID mice respond by upregulation of adhesion molecule expression when challenged in vivo with proinflammatory cytokines or activated PBMC.     

Molecular specialization of breast vasculature: a breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature

In vivo phage display identifies peptides that selectively home to the vasculature of individual organs, tissues, and tumors. Here we report the identification of a cyclic nonapeptide, CPGPEGAGC, which homes to normal breast tissue with a 100-fold selectivity over nontargeted phage. The homing of the phage is inhibited by its cognate synthetic peptide. Phage localization in tissue sections showed that the breast-homing phage binds to the blood vessels in the breast, but not in other tissues. The phage also bound to the vasculature of hyperplastic and malignant lesions in transgenic breast cancer mice. Expression cloning with a phage-displayed cDNA library yielded a phage that specifically bound to the breast-homing peptide. The cDNA insert was homologous to a fragment of aminopeptidase P. The homing peptide bound aminopeptidase P from malignant breast tissue in affinity chromatography. Antibodies against aminopeptidase P inhibited the in vitro binding of the phage-displayed cDNA to the peptide and the in vivo homing of phage carrying the peptide. These results indicate that aminopeptidase P is the receptor for the breast-homing peptide. This peptide may be useful in designing drugs for the prevention and treatment of breast cancer.     

The role of vascular cell adhesion molecule 1/ very late activation antigen 4 in endothelial progenitor cell recruitment to rheumatoid arthritis synovium

OBJECTIVE: Marrow-derived endothelial progenitor cells (EPCs) are important in the neovascularization that occurs in diverse conditions such as cardiovascular disorders, inflammatory diseases, and neoplasms. In rheumatoid arthritis (RA), synovial neovascularization propels disease by nourishing the inflamed and hyperproliferative synovium. This study was undertaken to investigate the hypothesis that EPCs selectively home to inflamed joint tissue and may perpetuate synovial neovascularization. METHODS: In a collagen-induced arthritis (CIA) model, neovascularization and EPC accumulation in mouse ankle synovium was measured. In an antibody-induced arthritis model, EPC recruitment to inflamed synovium was evaluated. In a chimeric SCID mouse/human synovial tissue (ST) model, mice were engrafted subcutaneously with human ST, and EPC homing to grafts was assessed 2 days later. EPC adhesion to RA fibroblasts and RA ST was evaluated in vitro. RESULTS: In mice with CIA, cells bearing EPC markers were significantly increased in peripheral blood and accumulated in inflamed synovial pannus. EPCs were 4-fold more numerous in inflamed synovium from mice with anti-type II collagen antibody-induced arthritis versus controls. In SCID mice, EPC homing to RA ST was 3-fold greater than to normal synovium. Antibody neutralization of vascular cell adhesion molecule 1 (VCAM-1) and its ligand component alpha4 integrin potently inhibited EPC adhesion to RA fibroblasts and RA ST cryosections. CONCLUSION: These data demonstrate the selective recruitment of EPCs to inflamed joint tissue. The VCAM-1/very late activation antigen 4 adhesive system critically mediates EPC adhesion to cultured RA fibroblasts and to RA ST cryosections. These findings provide evidence of a possible role of EPCs in the synovial neovascularization that is critical to RA pathogenesis.     

Targeting the prostate for destruction through a vascular address

Organ specific drug targeting was explored in mice as a possible alternative to surgery to treat prostate diseases. Peptides that specifically recognize the vasculature in the prostate were identified from phage-displayed peptide libraries by selecting for phage capable of homing into the prostate after an i.v. injection. One of the phage selected in this manner homed to the prostate 10-15 times more than to other organs. Unselected phage did not show this preference. The phage bound also to vasculature in the human prostate. The peptide displayed by the prostate-homing phage, SMSIARL (single letter code), was synthesized and shown to inhibit the homing of the phage when co-injected into mice with the phage. Systemic treatment of mice with a chimeric peptide consisting of the SMSIARL homing peptide, linked to a proapoptotic peptide that disrupts mitochondrial membranes, caused tissue destruction in the prostate, but not in other organs. The chimeric peptide delayed the development of the cancers in prostate cancer-prone transgenic mice (TRAMP mice). These results suggest that it may be possible to develop an alternative to surgical prostate resection and that such a treatment may also reduce future cancer risk.     

Cutaneous lymphocyte antigen-positive T lymphocytes preferentially migrate to the skin but not to the joint in psoriatic arthritis

Objective. To investigate whether T cell migration into different sites of inflammation (skin and synovium) within the same individual is principally regulated by tissue-specific homing or by more general mechanisms related to inflammation. Methods. Expression of cutaneous lymphocyte antigen (CLA) and its ligand, E-selectin, was analyzed by immunohistochemistry and immunofluorescence using paired skin and synovial membrane (SM) samples from patients with psoriatic arthritis (PsA). To investigate disease specificity, delayed-type hypersensitivity (DTH) skin lesions, induced by tuberculin purified protein derivative, and SM from patients with rheumatoid arthritis (RA), were studied as controls. To directly examine cell migration in vivo, the proportion of CLA+ T lymphocytes migrating into suction-induced skin blisters was assessed by flow cytometry. Using the same technique, levels of paired peripheral blood and synovial fluid (SF) T cells were also analyzed. Results. CLA+ T cells preferentially accumulated in the skin, but not in the joint, of patients with PsA. Similarly, CLA+ T lymphocytes predominated in the DTH skin lesions of RA patients, but were very rare in the SM of RA patients, and were scarcely represented in the SF of patients with several chronic inflammatory arthropathies. In addition, CLA+ T lymphocytes preferentially migrated into epidermal skin blisters. This preferential pattern of CLA+ T cell accumulation was not related to the selective expression of E-selectin, since this was similar in the skin and SM of both PsA and RA patients. Conclusion. The distinct pattern of T cell infiltration into sites of inflammation within the skin and synovium is regulated by both organ-specific homing and general inflammation-related mechanisms.     

Expression of sentrin, a novel antiapoptotic molecule, at sites of synovial invasion in rheumatoid arthritis

OBJECTIVE: Sentrin, a novel antiapoptotic molecule, has been shown to interact with the signal-competent form of Fas/APO-1 and tumor necrosis factor receptor I (TNFRI), and thereby, to protect cells against anti-Fas/APO-1- and TNF-induced cell death. Since reduced apoptosis in the synovial lining is supposed to contribute to synovial hyperplasia in rheumatoid arthritis (RA), we searched for the expression of sentrin-1 messenger RNA (mRNA) in synovium from patients with RA. METHODS: The expression of sentrin-1 mRNA was examined by in situ hybridization on snap-frozen sections of normal and RA synovial tissues as well as on paraffin-embedded RA synovial specimens, including the interface of cartilage-bone and invading synovium. Immunohistochemical double labeling after in situ hybridization was performed to further characterize sentrin-1 mRNA-expressing cells. In addition, quantitative analysis of sentrin-1 mRNA expression in RA synovial fibroblasts (RASF), osteoarthritis synovial fibroblasts (OASF), and normal fibroblasts was performed by quantitative real-time polymerase chain reaction. Expression levels were standardized to the expression of GAPDH. The in vivo maintenance of sentrin expression in RASF aggressively invading human cartilage was explored in the SCID mouse model of RA. RESULTS: A marked expression of sentrin-1 mRNA could be seen in all RA synovial specimens, predominantly in SF of the lining layer and at sites of invasion of RA synovium into cartilage. In normal synovial tissues, no sentrin-1 mRNA was detectable. RASF showed a maximum 32.5-fold (mean +/- SD 14.9 +/- 11.6) increase of sentrin-1 mRNA expression compared with normal fibroblasts and a maximum 31.4-fold (mean +/-SD 14.3 +/- 10.9) increase compared with OASF. When coimplanted with normal human cartilage in the SCID mouse model, invading RASF maintained their sentrin-1 mRNA expression for at least 60 days in vivo. CONCLUSION: The marked expression of sentrin in rheumatoid synovial tissue, but not in normal or OA synovial tissue, may contribute to the modulation of Fas- and TNFR-mediated apoptosis in RA synovium, and thereby extend the lifespan of invasive, cartilage-destructive SF.     

Identification of targeting peptides for ischemic myocardium by in vivo phage display

Therapies selectively targeting ischemic myocardium could be applied by intravenous injection. Here, we report an approach for ischemic tissue-selective targeting based on in vivo screening of random peptide sequences using phage display. We performed in vivo biopanning using a phage library in a rat model of ischemia-reperfusion and identified three peptide motifs, CSTSMLKAC, CKPGTSSYC, and CPDRSVNNC, that exhibited preferential binding to ischemic heart tissue compared to normal heart as well as other control organs. The CSTSMLKAC sequence was capable of mediating selective homing of phage to ischemic heart tissue. The CSTSMLKAC peptide was then made as a fusion protein with Sumo-mCherry and injected intravenously in a mouse model of myocardial ischemia-reperfusion injury; subsequently, bio-distribution of Sumo-mCherry-CSTSMLKAC was measured with quantitative ELISA. The targeting peptide led to a significant increase in homing to ischemic left ventricle compared to tissues from non-ischemic left ventricle, the right ventricle, lung, liver, spleen, skeletal muscle, and brain (all p < 0.001). These results indicate that the peptide sequence CSTSMLKAC represents a novel molecular tool that may be useful in targeting ischemic tissue and delivering bioengineered proteins into the injured myocardium by systemic intravenous administration.     

Ephrin A2 receptor targeting does not increase adenoviral pancreatic cancer transduction in vivo

AIM： To generate an adenoviral vector specifically targeting the EphA2 receptor （EphA2R） highly expressed on pancreatic cancer cells in vivo. METHODS： YSA, a small peptide ligand that binds the EphA2R with high affinity, was inserted into the HI loop of the adenovirus serotype 5 fiber knob. To further increase the specificity of this vector, binding sites for native adenoviral receptors, the coxsackie and adenovirus receptor （CAR） and integrin, were ablated from the viral capsid. The ablated retargeted adenoviral vector was produced on 293T cells. Specific targeting of this novel adenoviral vector to pancreatic cancer was investigated on established human pancreatic cancer cell lines. Upon demonstrating specific in vitro targeting, in vivo targeting to subcutaneous growing human pancreatic cancer was tested by intravenous and intraperitoneal administration of the ablated adenoviral vector. RESULTS： Ablation of native cellular binding sites reduced adenoviral transduction at least 100-fold. Insertion of the YSA peptide in the HI loop restored adenoviral transduction of EphA2R-expressing cells but not of cells lacking this receptor. YSA-mediated transduction was inhibited by addition of synthetic YSA peptide. The transduction specificity of the ablated retargeted vector towards human pancreatic cancer cells was enhanced almost 10-fold in vitro. In a subsequent in vivo study in a nude （nu/nu） mouse model however, no increased adenoviral targeting to subcutaneously growing human pancreas cancer nodules was seen upon injection into the tail vein, nor upon injection into the peritoneum. CONCLUSION： Targeting the EphA2 receptor increases specificity of adenoviral transduction of human pancreatic cancer cells in vitro but fails to enhance pancreatic cancer transduction in vivo.     

Direct selection of a human antibody fragment directed against the tumor T-cell epitope HLA-A1-MAGE-A1 from a nonimmunized phage-Fab library

Antitumor antibodies with the same specificity as cytotoxic T lymphocytes that recognize antigenic peptides encoded by tumor-associated genes and presented by MHC class I molecules would be valuable tools to analyze the antigenicity or target tumor cells in vivo. To obtain a human antibody directed against a peptide encoded by gene melanoma-associated antigen (MAGE)-A1 and presented by HLA-A1 molecules, we selected a large phage Fab antibody repertoire on a recombinant version of the complex HLA-A1-MAGE-A1 produced by in vitro refolding. One of the selected phage antibodies shows binding to HLA-A1 complexed with the MAGE-A1 peptide, but does not show binding to HLA-A1 complexed with a peptide encoded by gene MAGE-A3 and differing from the MAGE-A1 peptide by only three residues. Phages carrying this recombinant antibody bind to HLA-A1(+) cells only after in vitro loading with MAGE-A1 peptide. These results indicate that nonimmunized phage Fab libraries are a source of antibodies with a T cell antigen receptor-like specificity. The human anti-HLA-A1-MAGE-A1 antibody described here may prove very useful for monitoring the cell surface expression of these complexes, and eventually, as a targeting reagent for the specific immunotherapy of HLA-A1 patients bearing a MAGE-A1-positive tumor.     

Tissue trafficking patterns of effector memory CD4+ T cells in rheumatoid arthritis

OBJECTIVE: Clonal populations of CD4+,CD28- T cells accumulating in rheumatoid arthritis functionally resemble end-differentiated, nondividing, short-lived effector memory cells that reside in peripheral tissues. We undertook this study to examine the tissue niche for CD4+,CD28- T cells and the signals regulating their survival and tissue homing patterns. METHODS: Chemokine receptor expression on CD4+,CD28- T cell clones and peripheral blood lymphocytes was assessed by multicolor cytometry. In vitro chemotaxis and transendothelial migration were examined in a Transwell system. In vivo tissue-homing patterns were established by adoptively transferring fluorescence-labeled T cell clones into SCID mice engrafted either with rheumatoid synovium or with human lymph nodes. RESULTS: CD4+,CD28- T cell clones adoptively transferred into human tissue-SCID mouse chimeras infiltrated rheumatoid synovium but preferentially homed to lymph nodes. Such T cells coexpressed the chemokine receptors CCR7, CCR5, and CXCR4 and migrated in response to both inflammatory chemokines (CCL5) and homing chemokines (CXCL12). T cell receptor crosslinking abrogated chemotactic responsiveness. In contrast, interleukin-12 stimulation induced the up-regulation of CCR5 and a shift in the in vivo homing pattern away from the lymph nodes toward the inflamed synovium. CONCLUSION: CD4+,CD28- T cells resemble both short-lived effector memory cells and long-lived central memory cells, and they find a niche both in inflamed synovium and in lymph nodes. Nonspecific cytokine stimulation, not antigen recognition, triggers the transition from the lymph node to the synovium. By maintaining CCR7 expression, these end-differentiated T cells can home to lymphoid organs, enhance their survival, support clonal expansion, and perpetuate autoreactivity.     

Expression of angiogenic factors in juvenile rheumatoid arthritis: correlation with revascularization of human synovium engrafted into SCID mice

OBJECTIVE: Although increased vascularity was noted in early histopathologic studies of juvenile rheumatoid arthritis (JRA) synovium, the available data on angiogenesis in JRA are very limited. The main purposes of this study were to assess expression of the key angiogenic factors in JRA synovium, and to evaluate a SCID mouse model of JRA as an approach to study in vivo regulation of the expression of these factors in JRA. METHODS: RNase protection assay was used to assess the expression of the key angiogenic factors in fresh JRA synovium and in JRA synovial tissue fragments that had been minced and then implanted into SCID mice. Vascularity of the samples was assessed by immunohistochemical staining for von Willebrand factor. Synovial specimens obtained from patients with osteoarthritis (OA) or other noninflammatory arthropathies were used as controls. RESULTS: Detectable levels of messenger RNA for vascular endothelial growth factor and angiopoietin 1 and their respective receptors, as well as endoglin and thrombin receptors, were present in all JRA tissue specimens studied. The levels of expression of these factors in JRA tissues were significantly higher than those in tissues obtained from patients with OA or other noninflammatory arthropathies. Furthermore, increased expression of the key angiogenic factors in the fresh JRA tissues correlated with the exuberant revascularization of JRA minced tissue fragments implanted into SCID mice. This was in sharp contrast to the poor revascularization of implanted OA tissues. CONCLUSION: JRA synovium is characterized by high angiogenic activity. SCID mouse-human JRA synovium chimeras may provide a good approach to study the in vivo regulation of angiogenesis in JRA.     

Using shape effects to target antibody-coated nanoparticles to lung and brain endothelium

Vascular endothelium offers a variety of therapeutic targets for the treatment of cancer, cardiovascular diseases, inflammation, and oxidative stress. Significant research has been focused on developing agents to target the endothelium in diseased tissues. This includes identification of antibodies against adhesion molecules and neovascular expression markers or peptides discovered using phage display. Such targeting molecules also have been used to deliver nanoparticles to the endothelium of the diseased tissue. Here we report, based on in vitro and in vivo studies, that the specificity of endothelial targeting can be enhanced further by engineering the shape of ligand-displaying nanoparticles. In vitro studies performed using microfluidic systems that mimic the vasculature (synthetic microvascular networks) showed that rod-shaped nanoparticles exhibit higher specific and lower nonspecific accumulation under flow at the target compared with their spherical counterparts. Mathematical modeling of particle–surface interactions suggests that the higher avidity and specificity of nanorods originate from the balance of polyvalent interactions that favor adhesion and entropic losses as well as shear-induced detachment that reduce binding. In vivo experiments in mice confirmed that shape-induced enhancement of vascular targeting is also observed under physiological conditions in lungs and brain for nanoparticles displaying anti–intracellular adhesion molecule 1 and anti-transferrin receptor antibodies.     

Peptides selected for binding to clotted plasma accumulate in tumor stroma and wounds

Screening of a phage library for peptides that bind to clotted plasma in the presence of liquid plasma yielded two cyclic decapeptides, CGLIIQKNEC (CLT1) and CNAGESSKNC (CLT2). When injected intravenously into mice bearing various types of tumors, fluorescein-conjugated CLT peptides accumulated in a fibrillar meshwork in the extracellular compartment of the tumors, but were not detectable in other tissues of the tumor-bearing mice. The tumor homing of both peptides was strongly reduced after coinjection with unlabeled CLT2, indicating that the two peptides recognize the same binding site. The CLT peptide fluorescence colocalized with staining for fibrin(ogen) present in the extravascular compartment of tumors, but not in other tissues. The CLT peptides did not home to tumors grown in fibrinogen-null mice or in mice that lack plasma fibronectin. The CLT peptides also accumulated at the sites of injury in arteries, skeletal muscle, and skin. We conclude that the CLT peptides recognize fibrin-fibronectin complexes formed by clotting of plasma proteins that have leaked into the extravascular space in tumors and other lesions. These peptides may be useful in targeting diagnostic and therapeutic materials into tumors and injured tissues.     

Identification of peptides targeting the surface of Plasmodium falciparum-infected erythrocytes using a phage display peptide library

Drastic changes in the plasma membrane of Plasmodium falciparum-infected red blood cells (iRBCs) make the surface of iRBCs distinct from that of the uninfected erythrocyte. To identify small peptides that would specifically recognize the altered surface of iRBCs, we screened a phage display peptide library (PDL) on the surface of iRBCs. After the sixth panning of the PDL, eight phage clones of 18 sequenced clones had the same sequence, LVDAAAL (named P1) and specific binding of P1 to the surface of iRBCs was confirmed using phage expressing P1 peptides and synthetic P1 peptide. When P1 peptide was conjugated with a peptide having moderate hemolytic activity, the peptide conjugate inhibited the growth of intracellular parasites in a dose-dependent manner, whereas control peptides were without effect. Our results demonstrate that the P1 peptide may be a lead compound for the development of anti-malarial agents targeting the surface of iRBCs.     

Stromal cell-derived factor 1 (CXCL12) induces monocyte migration into human synovium transplanted onto SCID Mice

OBJECTIVE: The mechanisms by which monocyte/macrophage cells migrate to the joint involve a series of integrated adhesion and signaling events in which chemokines and their receptors are strongly implicated. This study was undertaken to investigate the hypothesis that stromal cell-derived factor 1 (SDF-1), a CXC chemokine (CXCL12), plays a critical role in monocyte/macrophage localization to synovium. METHODS: SDF-1 and CXC receptor 4 (CXCR4) expression in rheumatoid arthritis (RA) and osteoarthritis synovium and graft SDF-1, tumor necrosis factor alpha (TNF alpha), and human and murine vascular markers were examined by immunohistochemistry and double-immunofluorescence. The functional capacity of SDF-1 to modulate monocyte migration into joints was investigated by examining the localization of pro-myelomonocytic U937 cells into synovial tissue transplanted into SCID mice. SDF-1, TNF alpha, or saline was injected into graft sites and response determined by the number of fluorescently labeled U937 cells (injected intravenously) detected in grafts by ultraviolet microscopy. RESULTS: SDF-1 and CXCR4 were highly expressed in CD68+ cells in the RA synovium. SDF-1 induced U937 cell migration in vitro and in vivo in a dose-dependent manner and, in vivo, SDF-1 was more effective than TNF alpha. In contrast to TNF alpha, SDF-1 did not induce intracellular adhesion molecule 1 in transplant microvasculature. Furthermore, intragraft injection of SDF-1 did not up-regulate TNF alpha, or vice versa. CONCLUSION: This study demonstrates, for the first time, that SDF-1 is functional in vivo when injected into synovial grafts. In addition, SDF-1 is more potent than TNF alpha, and its mechanisms of action appear to be autonomous. Therefore, SDF-1 may be an important TNF-independent molecule involved in the migration to and retention of inflammatory effector cells in the joint.     

A fragment of the HMGN2 protein homes to the nuclei of tumor cells and tumor endothelial cells in vivo

We used a screening procedure to identify protein domains from phage-displayed cDNA libraries that bind both to bone marrow endothelial progenitor cells and tumor vasculature. Screening phage for binding of progenitor cell-enriched bone marrow cells in vitro, and for homing to HL-60 human leukemia cell xenograft tumors in vivo, yielded a cDNA fragment that encodes an N-terminal fragment of human high mobility group protein 2 (HMGN2, formerly HMG-17). Upon i.v. injection, phage displaying this HMGN2 fragment homed to HL-60 and MDA-MB-435 tumors. Testing of subfragments localized the full binding activity to a 31-aa peptide (F3) in the HMGN2 sequence. Fluorescein-labeled F3 peptide bound to and was internalized by HL-60 cells and human MDA-MB-435 breast cancer cells, appearing initially in the cytoplasm and then in the nuclei of these cells. Fluorescent F3 accumulated in HL-60 and MDA-MB-435 tumors after an i.v. injection, appearing in the nuclei of tumor endothelial cells and tumor cells. Thus, F3 can carry a payload (phage, fluorescein) to a tumor and into the cell nuclei in the tumor. This peptide may be suitable for targeting cytotoxic drugs and gene therapy vectors into tumors.     

A new model for rheumatoid arthritis generated by engraftment of rheumatoid synovial tissue and normal human cartilage into scid mice

Objective. A new animal model was used to study the interaction between rheumatoid synovial cells and cartilage and to explore the cellular basis of rheumatoid joint destruction. Methods. Fresh synovial tissue derived from patients with rheumatoid arthritis was implanted with normal human cartilage into SCID mice, either subcutaneously or under the renal capsule, for up to 304 days. The implants were analyzed by light and electron microscopy, as well as by immunohistochemistry and in situ hybridization. Results. Human synovial tissue and cartilage implanted in SCID mice are maintained by the animals for up to 304 days. After 35 days, focal erosions occur at the site of attachment of synovial lining cells to the cartilage. After 105 days, a pannus-like formation, consisting of proliferating synovial fibroblast-like cells invading the cartilage, is observed. The fibroblast nature of these cells was supported by observation of only focal expression of the macrophage markers CD14 and CD68. Cells at the immediate site of cartilage destruction express messenger RNA for cathepsin L, whereas cathepsin D messenger RNA was detected in subsynovial regions away from the site of destruction. The human origin of the tissue involved in cartilage destruction was demonstrated using monoclonal antibodies to HLA-ABC and human type IV collagen. Conclusion. The present approach introduces a novel in vivo model of rheumatoid arthritis for the study of the molecular and cellular mechanisms of rheumatoid joint destruction at sites of synovial attachment to cartilage. In this model, the SCID mouse acts as a useful host for studying the properties of rheumatoid synovium in the absence of circulating human blood components.