The activating receptor NKp46 is essential for the development of type 1 diabetes.

The mechanism of action of natural killer (NK) cells in type 1 diabetes is still unknown. Here we show that the activating receptor NKp46 recognizes mouse and human ligands on pancreatic beta cells. NK cells appeared in the pancreas when insulitis progressed to type 1 diabetes, and NKp46 engagement by beta cells led to degranulation of NK cells. NKp46-deficient mice had less development of type 1 diabetes induced by injection of a low dose of streptozotocin. Injection of soluble NKp46 proteins into nonobese diabetic mice during the early phase of insulitis and the prediabetic stage prevented the development of type 1 diabetes. Our findings demonstrate that NKp46 is essential for the development of type 1 diabetes and highlight potential new therapeutic modalities for this disease.

In Vitro and in Vivo Characterization of MOD-4023, a Long-Acting Carboxy-Terminal Peptide (CTP)-Modified Human Growth Hormone.

MOD-4023 is a novel long-acting version of human growth hormone (hGH), containing the carboxy-terminal peptide (CTP) of human chorionic gonadotropin (hCG). MOD-4023 is being developed as a treatment for adults and children with growth hormone deficiency (GHD), which would require fewer injections than currently available GH formulations and thus reduce patient discomfort and increase compliance. This study characterizes MOD-4023's binding affinities for the growth hormone receptor, as well as the pharmacokinetic and pharmacodynamics, toxicology, and safety profiles of repeated dosing of MOD-4023 in Sprague-Dawley rats and Rhesus monkeys. Although MOD-4023 exhibited reduced in vitro potency and lower affinity to the GH receptor than recombinant hGH (rhGH), administration of MOD-4023 every 5 days in rats and monkeys resulted in exposure comparable to daily rhGH, and the serum half-life of MOD-4023 was significantly longer. Repeated administration of MOD-4023 led to elevated levels of insulin-like growth factor 1 (IGF-1), and twice-weekly injections of MOD-4023 resulted in larger increase in weight gain with fewer injections and a lower accumulative hGH dose. Thus, the increased half-life of MOD-4023 in comparison to hGH may increase the frequency of protein-receptor interactions and compensate for its decreased in vitro potency. MOD-4023 was found to be well-tolerated in rats and monkeys, with minimal adverse events, suggesting an acceptable safety profile. These results provide a basis for the continued clinical development of MOD-4023 as a novel treatment of GHD in children and adults.

Genome-wide siRNA screen reveals a new cellular partner of NK cell receptor KIR2DL4: heparan sulfate directly modulates KIR2DL4-mediated responses.

KIR2DL4 (CD158d) is a distinct member of the killer cell Ig-like receptor (KIR) family in human NK cells that can induce cytokine production and cytolytic activity in resting NK cells. Soluble HLA-G, normally expressed only by fetal-derived trophoblast cells, was reported to be a ligand for KIR2DL4; however, KIR2DL4 expression is not restricted to the placenta and can be found in CD56(high) subset of peripheral blood NK cells. We demonstrated that KIR2DL4 can interact with alternative ligand(s), expressed by cells of epithelial or fibroblast origin. A genome-wide high-throughput siRNA screen revealed that KIR2DL4 recognition of cell-surface ligand(s) is directly regulated by heparan sulfate (HS) glucosamine 3-O-sulfotransferase 3B1 (HS3ST3B1). KIR2DL4 was found to directly interact with HS/heparin, and the D0 domain of KIR2DL4 was essential for this interaction. Accordingly, exogenous HS/heparin can regulate cytokine production by KIR2DL4-expressing NK cells and HEK293T cells (HEK293T-2DL4), and induces differential localization of KIR2DL4 to rab5(+) and rab7(+) endosomes, thus leading to downregulation of cytokine production and degradation of the receptor. Furthermore, we showed that intimate interaction of syndecan-4 (SDC4) HS proteoglycan (HSPG) and KIR2DL4 directly affects receptor endocytosis and membrane trafficking.

Safety of Repeated Administration of Xenogeneic Human Apoptotic State (Allocetra-OTS) in Sprague Dawley Rats.

Apoptotic cells possess immunomodulatory effects that can be utilized to treat imbalanced immune conditions. Information on the preclinical safety of such treatment is sparse. In this study, the safety of apoptotic cells (Allocetra-OTS) was assessed in a GLP toxicological study on Sprague Dawley rats. Three doses of Allocetra-OTS or vehicle were administered intravenously (IV) for 3 consecutive days. Animals in the main study were sacrificed on day 4, while animals from the recovery groups were kept for 14 or 28 days. Allocetra-OTS was well tolerated, and no adverse effects were observed in terms of body weight, clinical signs, food consumption, or ophthalmologic observation. Thus, the No Observed Adverse Effect Level (NOAEL) dose was determined as the highest dose administered. An observed elevation in immune cells was suspected to be due to Allocetra-OTS, similarly to other clinical chemistry parameters; however, it was resolved in the recovery phases. Splenomegaly and dose-related extramedullary hematopoiesis (EMH) in the red pulp were observed, with no adverse events, and were considered to be a normal and expected reaction following the IV administration of cell-based therapies. In conclusion, under the conditions of this study, Allocetra-OTS was concluded to be safe, further supporting its potential candidacy for clinical studies.

Apoptotic cells for treatment of acute respiratory distress syndrome associated with COVID-19.

Background: Hyper-inflammatory immune response, a hallmark of severe COVID-19, is associated with increased mortality. Acute respiratory distress syndrome (ARDS) is a common manifestation. We undertook two phase I/II studies in five and then 16 subjects with severe/critical COVID-19 to assess the safety and preliminary efficacy of apoptotic cells (Allocetra™-OTS, Enlivex Therapeutics), a cellular immunomodulatory therapy that reprograms macrophages to reduce hyper-inflammatory response severity. Methods: Eligible patients presenting to the Emergency Room with severe COVID-19 and respiratory dysfunction received one intravenous administration of Allocetra™-OTS and were monitored for adverse events (AEs) for 28 days. The primary aim was to determine the safety profile of treatment; secondary aims were recovery from ARDS, intensive care unit (ICU) and hospital length-of-stay, and mortality. Immune modulator markers were measured to elucidate the mechanism of action of Allocetra™-OTS. Results: 21 patients with severe-critical COVID-19 of Gamma, Alpha and Delta variants, were treated with a single dose of apoptotic cells. 19/21 patients had mild-to-severe ARDS at presentation. Median age was 53 years, 16/21 were males, 16/21 were overweight/obese. No serious related adverse events (SAEs) were reported. All 21 study subjects survived to day 28 (end of study); 19/21 recovered completely. Comparable mortality rates at the hospital were 3.8%-8.9% for age- and gender-matched patients, and 39%-55% for critical patients. Recovering patients exhibited rapid ARDS resolution and parallel resolution of inflammation markers and elevated cytokines/chemokines. Conclusion: In patients with severe/critical COVID-19 associated with ARDS, Allocetra™-OTS was safe, well-tolerated, and showed promising results for resolution of respiratory failure and inflammation. Trial registration: https://clinicaltrials.gov/ct2/show/study/NCT04513470, https://clinicaltrials.gov/ct2/show/study/NCT04590053, Identifiers NCT04513470, NCT04590053.

NKp44 receptor mediates interaction of the envelope glycoproteins from the West Nile and dengue viruses with NK cells.

Dengue virus (DV) and West Nile virus (WNV) have become a global concern due to their widespread distribution and their ability to cause a variety of human diseases. Antiviral immune defenses involve NK cells. In the present study, we investigated the interaction between NK cells and these two flaviviruses. We show that the NK-activating receptor NKp44 is involved in virally mediated NK activation through direct interaction with the flavivirus envelope protein. Recombinant NKp44 directly binds to purified DV and WNV envelope proteins and specifically to domain III of WNV envelope protein; it also binds to WNV virus-like particles. These WNV-virus-like particles and WNV-domain III of WNV envelope protein directly bind NK cells expressing high levels of NKp44. Functionally, interaction of NK cells with infective and inactivated WNV results in NKp44-mediated NK degranulation. Finally, WNV infection of cells results in increased binding of rNKp44 that is specifically inhibited by anti-WNV serum. WNV-infected target cells induce IFN-gamma secretion and augmented lysis by NKp44-expressing primary NK cells that are blocked by anti-NKp44 Abs. Our findings show that triggering of NK cells by flavivirus is mediated by interaction of NKp44 with the flavivirus envelope protein.

Dengue virus replicon expressing the nonstructural proteins suffices to enhance membrane expression of HLA class I and inhibit lysis by human NK cells.

Many viruses escape the cellular immune response by downregulating cell surface expression of major histocompatibility complex (MHC) class I molecules. However, infection of cells with flaviviruses can upregulate the expression of these molecules. In this study we analyzed the expression of MHC class I in K562 and THP-1 human cell lines that were stably transfected with self-replicating subgenomic dengue virus RNA (replicons) and express all the dengue virus nonstructural proteins together. We show that MHC class I expression is upregulated in the dengue virus replicon-expressing cells and that the binding of natural killer (NK) inhibitory receptors to these cells is augmented. This upregulation results in reduced susceptibility of the dengue virus replicon-expressing cells to NK lysis, indicating a possible mechanism for evasion of the dengue virus from NK cell recognition. Visualizing MHC class I expression in replicon-containing K562 and THP-1 cells by confocal microscopy demonstrated aggregation of MHC class I molecules on the cell surface. Finally, replicon-expressing K562 cells manifested increased TAP (transporter associated with antigen processing) and LMP (low-molecular-mass protein) gene transcription, while replicon-expressing THP-1 cells manifested increased NF-kappaB activity and MHC class I transcription. We suggest that expression of dengue virus nonstructural proteins is sufficient to induce MHC class I upregulation through both TAP-dependent and -independent mechanisms. Additionally, aggregation of MHC class I molecules on the cell membrane also contributes to significantly higher binding of low-affinity NK inhibitory receptors, resulting in lower sensitivity to lysis by NK cells.

H5-type influenza virus hemagglutinin is functionally recognized by the natural killer-activating receptor NKp44.

Antiviral immune defenses involve natural killer (NK) cells. We previously showed that the NK-activating receptor NKp44 is involved in the functional recognition of H1-type influenza virus strains by NK cells. In the present study, we investigated the interaction of NKp44 and the hemagglutinin of a primary influenza virus H5N1 isolate. Here we show that recombinant NKp44 recognizes H5-expressing cells and specifically interacts with soluble H5 hemagglutinin. H5-pseudotyped lentiviral particles bind to NK cells expressing NKp44. Following interaction with target cells expressing H5, pseudotyped lentiviral particles, or membrane-associated H5, NK cells show NKp44-mediated induced activity. These findings indicate that NKp44-H5 interactions induce functional NK activation.

Altered glycosylation of recombinant NKp30 hampers binding to heparan sulfate: a lesson for the use of recombinant immunoreceptors as an immunological tool.

NKp30 is a natural cytotoxicity receptor expressed by human NK cells and involved in NK lytic activity. We previously published that membranal heparan sulfate serves as a coligand for human NKp30. In the present study, we complement our results by showing direct binding of recombinant NKp30 to immobilized heparin. The heparan sulfate epitope(s) on target tumor cells and the heparin epitope(s) recognized by NKp30 share similar characteristics. Warren and colleagues (Warren HS, Jones AL, Freeman C, Bettadapura J, Parish CR. 2005. Evidence that the cellular ligand for the human NK cell activation receptor NKp30 is not a heparan sulfate glycosaminoglycan. J Immunol. 175:207-212) published that NKp30 does not bind to membranal heparan sulfate on target cells and that heparan sulfate is not involved in NKp30-mediated lysis. In the current study, we examine the binding of six different recombinant NKp30s to membranal heparan sulfate and conclude that NKp30 does interact with membranal heparan sulfate. Yet, two of the six recombinant NKp30s, including the commercially available recombinant NKp30 (employed by Warren et al.) did not show heparan sulfate-dependent binding. We demonstrate that this is due to an altered glycosylation of these two recombinant NKp30s. Upon removal of its N-linked glycans, heparan sulfate-dependent binding to tumor cells and direct binding to heparin were restored. Overall, our results emphasize the importance of proper glycosylation for analysis of NKp30 binding to its ligand and that membranal heparan sulfate could serve as a coligand for NKp30. At the cellular level, soluble heparan sulfate enhanced the secretion of IFNgamma by NK-92 natural killer cells activated with anti-NKp30 monoclonal antibody. We discuss the involvement of heparan sulfate binding to NKp30 in NKp30-mediated activation of NK cells.

Pharmacokinetics, Pharmacodynamics, and Safety of a Long-Acting Human Growth Hormone (MOD-4023) in Healthy Japanese and Caucasian Adults.

Daily injections of growth hormone (GH) as replacement therapy in GH-deficient (GHD) patients may cause poor compliance and inconvenience. C-terminal peptide-modified human GH (MOD-4023) has been developed for once-weekly administration in GHD adults and children. In the present study, the pharmacokinetics (PK) and pharmacodynamics (PD) of a single subcutaneous dose of MOD-4023 were evaluated in healthy Caucasian and Japanese adults, using a phase 1 double-blind, vehicle-controlled, randomized study design. The study was conducted in 42 healthy Japanese (n = 21) and Caucasian (n = 21) men receiving either MOD-4023 at a dose of 2.5, 7.5, or 15 mg or vehicle. In the 2.5- and 7.5-mg cohorts, no differences in mean MOD-4023 serum concentration were found between Japanese and Caucasian subjects. A comparison of PK parameters in the 15-mg group suggests a slower absorption rate of MOD-4023 in Japanese subjects. PD analysis showed no apparent differences in IGF-1 and IGFBP-3 plasma concentrations between the Japanese and Caucasian subjects and indicated that a dose of 15 mg achieved the maximal effect in both ethnic groups. MOD-4023 demonstrated a favorable safety profile and local tolerance following single-dose subcutaneous administration. This study provides additional support for the development of MOD-4023 as a long-acting human growth hormone formulation for once-weekly administration.

Long-Acting C-Terminal Peptide-Modified hGH (MOD-4023): Results of a Safety and Dose-Finding Study in GHD Children.

Context: Daily injections are required for growth hormone (GH) replacement therapy, which may cause low compliance as a result of inconvenience and distress in patients. Objective: C-terminal peptide-modified human GH (MOD-4023) is developed for once-a-week dosing regimen in GH-deficient (GHD) adults and children. The present trial was a safety and dose-finding study for weekly MOD-4023 in GHD children. Design: A multicenter, open-label, randomized, controlled phase 2 study in children with GHD, evaluating the safety, tolerability, pharmacokinetics/pharmacodynamics, and efficacy of three different weekly MOD-4023 doses, compared with daily recombinant human GH (r-hGH). Setting: The trial was conducted in 14 endocrinology centers in Europe. Patients: Fifty-three prepubertal children with GHD completed 12 months of treatment with either MOD-4023 (N = 42) or r-hGH (N = 11). Interventions: C-terminal peptide-modified hGH (MOD-4023) was administered weekly at a dose of either 0.25, 0.48, or 0.66 mg/kg/wk and compared with daily hGH at a dose of 0.24 mg/kg/wk. Results: MOD-4023 showed an estimated half-life approximately fivefold to 10-fold longer when compared with daily r-hGH. Insulin-like growth factor (IGF)-I and IGF-binding peptide 3 showed a dose-dependent increase during MOD-4023 treatment. IGF-I standard deviation score for MOD-4023 did not exceed +2. All MOD-4023 cohorts demonstrated adequate catch-up growth. The 0.66 mg/kg/wk dose demonstrated efficacy closest to daily r-hGH. No serious adverse events were observed during MOD-4023 treatment, and its tolerability was consistent with known properties of r-hGH. Conclusions: This study confirms the long-acting properties of MOD-4023 and shows a promising safety and tolerability profile. This provides support for initiation of a phase 3 study in GHD children using a single weekly injection of MOD-4023.

MOD-4023, a long-acting carboxy-terminal peptide-modified human growth hormone: results of a Phase 2 study in growth hormone-deficient adults.

OBJECTIVE: Growth hormone (GH) replacement therapy currently requires daily injections, which may cause distress and low compliance. C-terminal peptide (CTP)-modified growth hormone (MOD-4023) is being developed as a once-weekly dosing regimen in patients with GH deficiency (GHD). This study's objective is to evaluate the safety, pharmacokinetics (PK), pharmacodynamics (PD) and efficacy of MOD-4023 administered once-weekly in GHD adults. DESIGN: 54 adults with GHD currently treated with daily GH were normalized and randomized into 4 weekly dosing cohorts of MOD-4023 at 18.5%, 37%, 55.5% or 123.4% of individual cumulative weekly molar hGH dose. The study included 2 stages: Stage A assessed the effectiveness and PK/PD profiles of the 4 dosing regimens of MOD-4023. Stage B was an extension period of once-weekly MOD-4023 administration (61.7% molar hGH content) to collect further safety data and confirm the results from Stage A. RESULTS: Dose-dependent response was observed for both PK and PD data of weekly MOD-4023 treatment. Insulin-like growth factor I (IGF-I) SDS levels were maintained within normal range. The 18.5% cohort was discontinued due to low efficacy. MOD-4023 was well tolerated and exhibited favorable safety profile in all dose cohorts. The reported adverse events were consistent with known GH-related side effects. CONCLUSIONS: Once-weekly MOD-4023 administration in GHD adults was found to be clinically effective while maintaining a favorable safety profile and may obviate the need for daily injections. Weekly GH injections may improve compliance and overall outcome. The promising results achieved in this Phase 2 study led to a pivotal Phase 3 trial, which is currently ongoing.

Upregulation of MHC class I expression following dengue virus infection: the mechanism at the promoter level.

Unlike many other viruses that downregulate MHC class I expression on infected cell membranes, flaviviruses were reported to upregulate the MHC class I expression. Dengue virus was shown to induce HLA class I expression; however, the precise transcriptional mechanism that is used by the virus remains unclear. This article assessed the findings of a recently published report describing the mechanism used by dengue virus to induce HLA-A2 expression and characterizing the transcription factors that are involved. The study showed that p50/p65 and p65/65 NF-κB dimers bind to the class I regulatory complex within the HLA-A2 promoter. This finding and its significance for the design of possible antiviral therapeutic agents are discussed in this article.

Generating NK cell receptor-Fc chimera proteins from 293T cells and considerations of appropriate glycosylation.

The use of recombinant receptors as a scientific tool has become widespread in many research fields. Of particular interest are the natural killer (NK) receptors that play a major role in the immune response against tumors and virus-infected cells. We present here (i) a detailed protocol for the production and purification of soluble recombinant NK cell receptors tagged with human IgG1-Fc (thus termed receptor-Fc chimera or receptor-Ig fusion protein) and (ii) a protocol for cell staining with these recombinant receptor-Fc chimeras. As these recombinant proteins are produced in eukaryotic cells, we further discuss the glycosylation pattern of these receptors that might interfere with their ligand-binding phenotype.

Natural cytotoxicity receptors NKp30, NKp44 and NKp46 bind to different heparan sulfate/heparin sequences.

Natural Killer (NK) cells recognize and destroy tumors and virus-infected cells in an antibody-independent manner. The regulation of NK cells is mediated by activating and inhibiting receptors on the NK cell surface. One important family of activating receptors is the natural cytotoxicity receptors (NCRs) which include NKp30, NKp44 and NKp46. The NCRs initiate tumor targeting by recognition of heparan sulfate on cancer cells. This study aims to elucidate heparan sulfate structural motifs that are important for NCR binding. Microarray and surface plasmon resonance experiments with a small library of heparan sulfate/heparin oligosaccharides helped to clarify the binding preferences of the three NCRs. We demonstrate that the NCRs interact with highly charged HS/heparin structures, but differ in preferred modification patterns and chain lengths. The affinity of NKp30 and NKp44 for synthetic HS/heparin is approximately one order of magnitude higher than the affinity of NKp46. We further show the relevance of synthetic HS/heparin for the binding of NCRs to tumor cells and for NCR-mediated activation of natural killer cells. In conclusion, NCRs recognize different microdomains on heparan sulfate with different affinities.

Expression of ligands to NKp46 in benign and malignant melanocytes.

Human melanoma cell lines were shown to express ligands for the natural cytotoxicity receptor, NKp46, expressed by natural killer (NK) cells. We aimed to examine the expression of ligands for NKp46 by various primary human melanocytic cells and melanocytic lesions. Sections from primary nevi and melanomas were tested for expression of NKp46 ligands employing chimeric NKp46-Fc for staining. The melanocytes present in the reticular dermis were negative for NKp46 ligands in common nevi; in malignant melanocytic lesions, the deeper melanocytes were focally positive. In dermoepidermal junction of all melanocytic lesions, the melanocytes showed enhanced expression of NKp46 ligands. Melanophages in all lesions were consistently positive for NKp46 ligands. These observations establish the expression of NKp46 ligands by primary-transformed melanocytes. Normal melanocytes did not express ligands to NKp46. Therefore, the results show (i) a correlation between the malignant potential of the lesion and the expression of NKp46 ligands in the reticular dermis, and (ii) enhanced expression of NKp46 ligands in the active proliferation zone (dermoepidermal junction) of nevi and melanomas. Ligands to NKp46 were expressed on the membrane and within the cells. The physiological role of NKp46 ligands in the progression of malignancy within melanocytic lesions should be explored further.

Characterization of the recognition of tumor cells by the natural cytotoxicity receptor, NKp44.

NKp44 is a natural cytotoxicity receptor expressed by human NK cells upon activation. In this study, we demonstrate that cell surface heparan sulfate proteoglycans (HSPGs), expressed by target cells, are involved in the recognition of tumor cells by NKp44. NKp44 showed heparan sulfate-dependent binding to tumor cells; this binding was partially blocked with an antibody to heparan sulfate. In addition, direct binding of NKp44 to heparin was observed, and soluble heparin/heparan sulfate enhanced the secretion of IFNgamma by NK92 cells activated with anti-NKp44 monoclonal antibody. Basic amino acids, predicted to constitute the putative heparin/heparan sulfate binding site of NKp44, were mutated. Tumor cell recognition of the mutated NKp44 proteins was significantly reduced and correlated with their lower recognition of heparin. We previously reported that NKp44 recognizes the hemagglutinin of influenza virus (IV). Nevertheless, the ability of the mutated NKp44 proteins to bind viral hemagglutinin expressed by IV-infected cells was not affected. Thus, we suggest that heparan sulfate epitope(s) are ligands/co-ligands of NKp44 and are involved in its tumor recognition ability.

Characterization of the heparin/heparan sulfate binding site of the natural cytotoxicity receptor NKp46.

NKp46 is a member of a group of receptors collectively termed natural cytotoxicity receptors (NCRs) that are expressed by natural killer (NK) cells. NCRs are capable of mediating direct killing of tumor and virus-infected cells by NK cells. We have recently shown that NKp46 recognizes the heparan sulfate moieties of membranal heparan sulfate proteoglycans (HSPGs), thus enabling lysis of tumor cells by NK cells. In the current study, we further examined the residues in NKp46 that may be involved in heparan sulfate binding on tumor cells. On the basis of both the electrostatic potential map and comparison to the heparin binding site on human fibronectin, we predicted a continuous region containing the basic amino acids K133, R136, H139, R142, and K146 to be involved in NKp46 binding to heparan sulfate. Mutating these amino acids on NKp46D2 to noncharged amino acids retained its virus binding capacity but reduced its binding to tumor cells with a 10-100 fold lower K(D) when tested for direct binding to heparin. The minimal length of the heparin/heparan sulfate epitope recognized by NKp46 was eight saccharides as predicted from the structure and proven by testing heparin oligomers. Testing selectively monodesulfated heparin oligomers emphasized the specific contributions of O-sulfation, N-sulfation, and N-acetylation to epitope recognition by NKp46. The characterization of heparan sulfate binding region in NKp46 offers further insight into the identity of the ligands for NKp46 and the interaction of NK and cancers.

Membrane-associated heparan sulfate proteoglycans are involved in the recognition of cellular targets by NKp30 and NKp46.

Lysis of virus-infected and tumor cells by NK cells is mediated via natural cytotoxicity receptors (NCRs). We have recently shown that the NKp44 and NKp46 NCRs, but not the NKp30, recognize viral hemagglutinins. In this study we explored the nature of the cellular ligands recognized by the NKp30 and NKp46 NCRs. We demonstrate that target cell surface heparan sulfate proteoglycans (HSPGs) are recognized by NKp30 and NKp46 and that 6-O-sulfation and N-acetylation state of the glucose building unit affect this recognition and lysis by NK cells. Tumor cells expressing cell surface heparanase, CHO cells lacking membranal heparan sulfate and glypican-1-suppressed pancreatic cancer cells manifest reduced recognition by NKp30 and NKp46 and are lysed to a lesser extent by NK cells. Our results are the first clue for the identity of the ligands for NKp30 and NKp46. Whether the ligands are particular HSPGs, unusual heparan sulfate epitopes, or a complex of HSPGs and either other protein or lipid moieties remains to be further explored.