Soluble factors from the notochordal-rich intervertebral disc inhibit endothelial cell invasion and vessel formation in the presence and absence of pro-inflammatory cytokines.

BACKGROUND: Chronic low back pain can be associated with the pathological ingrowth of blood vessels and nerves into intervertebral discs (IVDs). The notochord patterns the IVD during development and is a source of anti-angiogenic soluble factors such as Noggin and Chondroitin sulfate (CS). These factors may form the basis for a new minimally invasive strategy to target angiogenesis in the IVD. OBJECTIVE: To examine the anti-angiogenic potential of soluble factors from notochordal cells (NCs) and candidates Noggin and CS under healthy culture conditions and in the presence of pro-inflammatory mediators. DESIGN: NC conditioned media (NCCM) was generated from porcine NC-rich nucleus pulposus tissue. To assess the effects of NCCM, CS and Noggin on angiogenesis, cell invasion and tubular formation assays were performed using human umbilical vein endothelial cells (HUVECs) ± tumor necrosis factor alpha (TNFα [10 ng/ml]). vascular endothelial growth factor (VEGF)-A, MMP-7, interleukin-6 (IL-6) and IL-8 mRNA levels were assessed using qRT-PCR. RESULTS: NCCM (10 & 100%), CS (10 and 100 µg) and Noggin (10 and 100 ng) significantly decreased cell invasion of HUVECs with and without TNFα. NCCM 10% and Noggin 10 ng inhibited tubular formation with and without TNFα and CS 100 µg inhibited tubules in Basal conditions whereas CS 10 µg inhibited tubules with TNFα. NCCM significantly decreased VEGF-A, MMP-7 and IL-6 mRNA levels in HUVECs with and without TNFα. CS and Noggin had no effects on gene expression. CONCLUSIONS: We provide the first evidence that soluble factors from NCs can inhibit angiogenesis by suppressing VEGF signaling. Notochordal-derived ligands are a promising minimally invasive strategy targeting neurovascular ingrowth and pain in the degenerated IVD.

Notochordal cell-derived therapeutic strategies for discogenic back pain.

An understanding of the processes that occur during development of the intervertebral disk can help inform therapeutic strategies for discogenic pain. This article reviews the literature to identify candidates that are found in or derived from the notochord or notochordal cells and evaluates the theory that such factors could be isolated and used as biologics to target the structural disruption, inflammation, and neurovascular ingrowth often associated with discogenic back pain. A systematic review using PubMed was performed with a primary search using keywords "(notochordal OR notochord) And (nerves OR blood vessels OR SHH OR chondroitin sulfate OR notch OR CTGF) NOT chordoma." Secondary searches involved keywords associated with the intervertebral disk and pain. Several potential therapeutic candidates from the notochord and their possible targets were identified. Studies are needed to further identify candidates, explore mechanisms for effect, and to validate the theory that these candidates can promote structural restoration and limit or inhibit neurovascular ingrowth using in vivo studies.

Stress protein-induced immunosuppression: inhibition of cellular immune effector functions following overexpression of haem oxygenase (HSP 32).

This is the first report on suppression of immune effector functions following upregulation of heat shock protein 32 (HSP 32), known as haem oxygenase (HO-1). Here we evaluated the effect of cobalt-protoporphyrin (CoPP)-induced HO-1 expression on cell-mediated immune responses. Administration of CoPP to CBA mice resulted in overexpression of HO-1 in the spleen, liver and kidneys. In vitro measurements of T cell-mediated and NK-cell-mediated cytotoxicity in spleens from CoPP-treated animals demonstrated a severe suppression of their effector functions while administration of Zn-PP or vitamin B12 had no effect. Furthermore, CoPP therapy decreased the lymphoproliferative alloresponse and differentiation of cytotoxic T cells. Inhibition of proliferation appeared to be due to cell growth arrest with an increased number of cells staying in G0/G1 phase. Despite the suppressed proliferative response, IL-2 production in the MLR was not inhibited. In contrast, CoPP decreased the production of IL-10, IFN-gamma and TNF-alpha. In vivo, CoPP prolonged the survival of heterotopic heart allografts in mice. The immunosuppressive effects following CoPP-mediated upregulation of HO-1 were similar to those observed after peptide-mediated upregulation of HO-1. The results indicate that overexpression of HO results in the inhibition of several immune effector functions and thus provides an explanation for stress-induced immunosuppression.

Characterization and biological significance of immunosuppressive peptide D2702.75-84(E --> V) binding protein. Isolation of heme oxygenase-1.

This is the first report on peptidic inhibitors of heme oxygenase. Such peptides were originally developed from the immunomodulatory peptide 2702.75-84 which corresponds to amino acid residues 75 to 84 of the alpha1-helix of HLA-B2702 (2702.75-84) and has been shown to be immunosuppressive in vitro and in vivo. In vitro, 2702.75-84 inhibited cytotoxic T- and natural killer cell- mediated target cell lysis, and in vivo peptide therapy resulted in prolongation of heart and skin allograft survival in mice. The peptide was also shown to bind to heat shock protein 70. However, D-enantiomers of 2702.75-84 and derivatives thereof, while still being immunosuppressive, did not bind to heat shock protein 70. This study was designed to identify proteins binding to peptide D2702.75-84(E --> V) (rvnlrialry) consisting of D-amino acids. Compared with 2702.75-84 (RENLRIALRY), glutamic acid residue 76 (E) was replaced with valine (V). Affinity chromatography using immobilized D2702.75-84(E --> V) and mouse and human cell extracts, resulted in the isolation of heme oxygenase-1 (HO-1). Peptide D2702.75-84 inhibited HO activity in vitro in a dose dependent manner. Similar to what has been observed with other inhibitors of HO, administration of peptide into mice resulted in an up-regulation of HO-1 mRNA and protein, as well as enzyme activity in liver, spleen and kidney. Other peptides derived from 2702.75-84 with similar immunomodulatory activity displayed similar effects. In contrast, inactive derivatives of 2702.75-84 had no effect on HO activity. Therefore, the immunosuppressive effects of the described immunomodulatory peptides are similar to those of cobalt-protoporphyrin, a known up-regulator of HO-1. Our results suggest that HO-1 modulation may be a novel mechanism of immunomodulation.

Immunosuppression by D-isomers of HLA class I heavy chain (amino acid 75 to 84)-derived peptides is independent of binding to HSC70.

BACKGROUND: Peptides derived from the class I heavy chain were shown to modulate immune responses in vitro and in vivo. A peptide derived from HLA-B2702 (2702.75-84) inhibited differentiation of cytotoxic T cells as well as T cell and natural killer cell-mediated cytotoxicity in vitro. Peptide-mediated immunomodulation seemed to be independent of the MHC proteins expressed by responder and stimulator cells. In vivo studies in rodents demonstrated prolongation of heart and skin allograft survival after peptide therapy. Here, the correlation between the peptide's biological activity and its amino acid sequence was analyzed using peptides derived from amino acid 75-84 of several mouse, rat, and human MHC class I proteins as well as peptides with single amino acid substitutions in the 2702.75-84 sequence. METHODS: Peptides consisting of both L- and D-amino acids were tested for inhibition of murine and human T cell-mediated and lymphokine-activated killer cell-mediated cytotoxicity, binding to hsc70, and prolongation of heart allograft survival in vivo. RESULTS: Replacement of glutamic acid residue (E) at position 75 with valine (V) resulted in a peptide [2702.75-84(E>V)] with increased in vitro and in vivo activity but unchanged affinity for hsc70. Surprisingly, both L- and D-isomers of 2702.75-84 and 2702.75-84(E>V) inhibited cytotoxic cells in vitro and prolonged heart allograft survival in vivo. However, as expected, the peptides consisting of D-amino acids did not bind to hsc70. CONCLUSION: Assuming that both D- and L-isomers modulate immune responses by similar mechanisms, these results suggest that the peptides' effect is independent of binding to hsc70.

Monoclonal antibody to the HLA class I alpha 3 domain inhibits T cell activation and prolongs cardiac allograft survival in HLA-transgenic mice.

Antibodies recognizing MHC class I molecules expressed on the surface of T cells have been shown to inhibit T cell responses in vitro. These findings suggested that therapy with such an antibody may prevent rejection and promote graft acceptance. We therefore tested the effect of an anti-HLA class I alpha 3 domain antibody (TP25.99) in vivo using transgenic C57BL/6 mice expressing HLA-B2705. Flow cytometric analysis confirmed the binding of TP25.99 to normal human peripheral blood lymphocytes and to mouse spleen cells, bone marrow cells and thymocytes isolated from hemizygous (+/-) transgenic littermates but not from homozygous (-/-) littermates. TP25.99 inhibited OKT-3-induced, but not PMA+ionomycin-induced, proliferation of human peripheral blood lymphocyte as well as anti-CD3 or Con A-induced proliferation of HLA+ mouse T cells. Both intact monoclonal antibody TP25.99 and TP25.99 Fab inhibited T cell proliferation. Reduced proliferation was associated with suppressed production of interleukin-2 as measured by ELISA. The efficacy of TP25.99 Fab in vivo was evaluated in a heart allograft model. Antibody therapy of (H-2h, B2705+) transgenic recipients of allogeneic Balb/c (H-2d) heart grafts prolonged graft survival significantly (MST = 19.8 +/- 6.4, p = 0.003) compared to treated (H-2b, B2705-) (MST = 9.17 +/- 2.2) or untreated (H-2b, B2705+) (MST = 10.0 +/- 2.8) transgenic recipients. This demonstrates that immunomodulation through anti-HLA class I antibody therapy can lead to prolongation of graft survival.

Soluble HLA antigens and ELISA--a new technology for crossmatch testing.

A soluble HLA ELISA for the detection of donor specific anti-HLA class I IgG antibodies was developed and compared with microlymphocytotoxicity. Donor sHLA was prepared from donor blood or purified blood lymphocytes and captured onto monoclonal antibody coated ELISA plates. After incubation of captured HLA with test serum, bound IgG antibodies were detected using a peroxidase-conjugated anti-human IgG antibody. Serum samples from patients on waiting lists to receive kidney transplants were tested by lymphocytotoxicity (AHG protocol) and/or sHLA ELISA in four different laboratories using HLA preparations from eight organ donors. Concordant crossmatch results were obtained for 854 (99%) of 864 ELISA crossmatches. In contrast, concordant results were obtained for 234 (91%) of 256 lymphocytotoxicity crossmatches. Interlaboratory reproducibility of ELISA results was 99%. In contrast, interlaboratory reproducibility of lymphocytotoxicity assay results was 78%. Endpoint titrations of serum specimens containing anti-HLA antibodies demonstrated equivalent sensitivity of ELISA and AHG lymphocytotoxicity crossmatch and similar sensitivity of ELISA and flow cytometry crossmatch. Specimens tested positive by lymphocytotoxicity without DTT treatment but negative with DTT treatment were tested negative by ELISA. Comparison of lymphocytotoxicity and ELISA crossmatch results showed an agreement of 94%. This demonstrates that detection of anti-donor HLA class I antibodies by ELISA is a reliable alternative to microlymphocytotoxicity testing.

A synthetic dimeric HLA class I peptide inhibits T cell activity in vitro and prolongs allogeneic heart graft survival in a mouse model.

A peptide derived from the alpha 1 domain of the human HLA class I heavy chain (amino acids 75-84; B2702.75-84) has been shown to inhibit human cytotoxic T and NK cell activity in a non-allele-restricted manner. In vivo, this peptide prolonged skin allograft survival in a murine model. Here we demonstrate prolongation of heart allograft survival in mice and extend the characterization of the immunomodulatory activity of B2702.75-84. Similar to what has been observed with retrovirus-derived peptides, the inhibitory capability of this peptide was increased when bound to a carrier protein. An increased immunomodulatory activity was also observed with the dimeric peptide B2702.84-75-75-84 or the multimeric B2702.75-84.MAP. This peptide not only inhibited cytotoxic T and NK cells but also anti-CD3-induced T cell proliferation as well as a mixed lymphocyte reaction (MLR). Flow cytometric analysis of T cells harvested from anti-CD3-stimulated spleen cell culture in the presence of B2702.84-75-75-84 showed decreased expression of activation markers (CD25, ICAM-1, Pgp-1, CD69) compared with untreated control cultures. The superior activity of B2702.84-75-75-84 could also be demonstrated in vivo. Administration of B2702.84-75-75-84 prolonged the survival of B6 (H2b) hearts in CBA (H2k) recipients to 15 +/- 2.7 (P = 0.0002 vs. control) days compared with 11.4 +/- 2.6 (P = 0.01) days in B2702.75-84 treated animals and 7.5 +/- 1.1 days in untreated controls. Administration of control peptides had no significant effect on allograft survival. In combination with a subtherapeutic dose of cyclosporine, B2702.75-84 induced long-term graft survival in 60% of recipients.