Impaired retinal angiogenesis in diabetes: role of advanced glycation end products and galectin-3

Suppression of angiogenesis during diabetes is a recognized phenomenon but is less appreciated within the context of diabetic retinopathy. The current study has investigated regulation of retinal angiogenesis by diabetic serum and determined if advanced glycation end products (AGEs) could modulate this response, possibly via AGE-receptor interactions. A novel in vitro model of retinal angiogenesis was developed and the ability of diabetic sera to regulate this process was quantified. AGE-modified serum albumin was prepared according to a range of protocols, and these were also analyzed along with neutralization of the AGE receptors galectin-3 and RAGE. Retinal ischemia and neovascularization were also studied in a murine model of oxygen-induced proliferative retinopathy (OIR) in wild-type and galectin-3 knockout mice (gal3(-/-)) after perfusion of preformed AGEs. Serum from nondiabetic patients showed significantly more angiogenic potential than diabetic serum (P <0.0001) and within the diabetic group, poor glycemic control resulted in more AGEs but less angiogenic potential than tight control (P <0.01). AGE-modified albumin caused a dose-dependent inhibition of angiogenesis (P <0.001), and AGE receptor neutralization significantly reversed the AGE-mediated suppression of angiogenesis (P <0.01). AGE-treated wild-type mice showed a significant increase in inner retinal ischemia and a reduction in neovascularization compared with non-AGE controls (P <0.001). However, ablation of galectin-3 abolished the AGE-mediated increase in retinal ischemia and restored the neovascular response to that seen in controls. The data suggest a significant suppression of angiogenesis by the retinal microvasculature during diabetes and implicate AGEs and AGE-receptor interactions in its causation.

The human prion protein residue 129 polymorphism lies within a cluster of epitopes for T cell recognition.

T cell immune responses to central nervous system-derived and other self-antigens are commonly described in both healthy and autoimmune individuals. However, in the case of the human prion protein (PrP), it has been argued that immunologic tolerance is uncommonly robust. Although development of an effective vaccine for prion disease requires breaking of tolerance to PrP, the extent of immune tolerance to PrP and the identity of immunodominant regions of the protein have not previously been determined in humans. We analyzed PrP T cell epitopes both by using a predictive algorithm and by measuring functional immune responses from healthy donors. Interestingly, clusters of epitopes were focused around the area of the polymorphic residue 129, previously identified as an indicator of susceptibility to prion disease, and in the C-terminal region. Moreover, responses were seen to PrP peptide 121-134 containing methionine at position 129, whereas PrP 121-134 [129V] was not immunogenic. The residue 129 polymorphism was also associated with distinct patterns of cytokine response: PrP 128-141 [129M] inducing IL-4 and IL-6 production, which was not seen in response to PrP 128-141 [129V]. Our data suggest that the immunogenic regions of human PrP lie between residue 107 and the C-terminus and that, like with many other central nervous system antigens, healthy individuals carry responses to PrP within the T cell repertoire and yet do not experience deleterious autoimmune reactions.

Advanced glycation end products (AGEs) co-localize with AGE receptors in the retinal vasculature of diabetic and of AGE-infused rats

Advanced glycation end products (AGEs), formed from the nonenzymatic glycation of proteins and lipids with reducing sugars, have been implicated in many diabetic complications; however, their role in diabetic retinopathy remains largely unknown. Recent studies suggest that the cellular actions of AGEs may be mediated by AGE-specific receptors (AGE-R). We have examined the immunolocalization of AGEs and AGE-R components R1 and R2 in the retinal vasculature at 2, 4, and 8 months after STZ-induced diabetes as well as in nondiabetic rats infused with AGE bovine serum albumin for 2 weeks. Using polyclonal or monoclonal anti-AGE antibodies and polyclonal antibodies to recombinant AGE-R1 and AGE-R2, immunoreactivity (IR) was examined in the complete retinal vascular tree after isolation by trypsin digestion. After 2, 4, and 8 months of diabetes, there was a gradual increase in AGE IR in basement membrane. At 8 months, pericytes, smooth muscle cells, and endothelial cells of the retinal vessels showed dense intracellular AGE IR. AGE epitopes stained most intensely within pericytes and smooth muscle cells but less in basement membrane of AGE-infused rats compared with the diabetic group. Retinas from normal or bovine-serum-albumin-infused rats were largely negative for AGE IR. AGE-R1 and -R2 co-localized strongly with AGEs of vascular endothelial cells, pericytes, and smooth muscle cells of either normal, diabetic, or AGE-infused rat retinas, and this distribution did not vary with each condition. The data indicate that AGEs accumulate as a function of diabetes duration first within the basement membrane and then intracellularly, co-localizing with cellular AGE-Rs. Significant AGE deposits appear within the pericytes after long-term diabetes or acute challenge with AGE infusion conditions associated with pericyte damage. Co-localization of AGEs and AGE-Rs in retinal cells points to possible interactions of pathogenic significance.

Rotavirus enterotoxin NSP4 has mucosal adjuvant properties

Rotavirus nonstructural protein 4 (NSP4) is a protein with pleiotropic properties. It functions in rotavirus morphogenesis, pathogenesis, and is the first described viral enterotoxin. Since many bacterial toxins function as potent mucosal adjuvants, we evaluated whether baculovirus-expressed recombinant simian rotavirus SA11 NSP4 possesses adjuvant activity by co-administering NSP4 with keyhole limpet hemocyanin (KLH), tetanus toxoid (TT) or ovalbumin (OVA) as model antigens in mice. Following intranasal immunization, NSP4 significantly enhanced both systemic and mucosal immune responses to model immunogens, as compared to the control group, in an antigen-specific manner. Both full-length and a cleavage product of SA11 NSP4 had adjuvant activity, localizing this activity to the C-terminus of the protein. NSP4 forms from virulent and avirulent porcine rotavirus OSU strain, and SA11 NSP4 localized within a 2/6-virus-like particle (VLP) also exhibited adjuvant effects. These studies suggest that the rotavirus enterotoxin NSP4 can function as an adjuvant to enhance immune responses for a co-administered antigen.

Toxicity of mildly modified low-density lipoproteins to cultured retinal capillary endothelial cells and pericytes

To investigate the role of modified low-density lipoproteins (LDL) in the pathogenesis of diabetic retinopathy, we studied the cytotoxicity of normal and mildly modified human LDL to bovine retinal capillary endothelial cells and pericytes in vitro. Pooled LDL was incubated (in phosphate-buffered saline-EDTA, 3 days, 37 degrees C) under 1) nitrogen with additional chelating agents and 2) air, to prepare normal and minimally oxidized LDL, respectively. Similar conditions, but with the addition of 50 mM D-glucose, were used to prepare glycated and glycoxidized LDL. None of the LDL preparations was recognized by the macrophage scavenger receptor, confirming limited modification. Retinal capillary endothelial cells and pericytes were grown to confluence and then exposed for 2 or 3 days to serum-free medium (1% albumin) supplemented with normal or modified LDL (100 mg/l) or to serum-free medium alone. Cytotoxicity was assessed by cell counting (live and total cells) and by cell protein determination. Compared with normal LDL, modified LDL were cytotoxic to both cell types at both time points, causing highly significant decreases in live and total cell counts (P <0.001) (analysis of variance). Reductions in cell protein also were significant for pericytes at day 3 (P = 0.016) and of borderline significance for endothelial cells at day 2 (P = 0.05) and day 3 (P = 0.063). Cytotoxicity increased as follows: normal <glycated <or = minimally oxidized <glycoxidized LDL. We conclude that, in diabetes, mild modification of LDL resulting from separate or combined processes of glycation and oxidation may contribute to chronic retinal capillary injury and thus to the development of diabetic retinopathy.

Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction

The recognition of microbial pathogens by the innate immune system involves Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns. Different TLRs recognize different pathogen-associated molecular patterns, with TLR-4 mediating the response to lipopolysaccharide from Gram-negative bacteria. All TLRs have a Toll/IL-1 receptor (TIR) domain, which is responsible for signal transduction. MyD88 is one such protein that contains a TIR domain. It acts as an adapter, being involved in TLR-2, TLR-4 and TLR-9 signalling; however, our understanding of how TLR-4 signals is incomplete. Here we describe a protein, Mal (MyD88-adapter-like), which joins MyD88 as a cytoplasmic TIR-domain-containing protein in the human genome. Mal activates NF-kappaB, Jun amino-terminal kinase and extracellular signal-regulated kinase-1 and -2. Mal can form homodimers and can also form heterodimers with MyD88. Activation of NF-kappaB by Mal requires IRAK-2, but not IRAK, whereas MyD88 requires both IRAKs. Mal associates with IRAK-2 by means of its TIR domain. A dominant negative form of Mal inhibits NF-kappaB, which is activated by TLR-4 or lipopolysaccharide, but it does not inhibit NF-kappaB activation by IL-1RI or IL-18R. Mal associates with TLR-4. Mal is therefore an adapter in TLR-4 signal transduction.

GDF-15 contributes to proliferation and immune escape of malignant gliomas

Growth and differentiation factor (GDF)-15 is a member of the transforming growth factor (TGF)-beta family. GDF-15 is necessary for the maintenance of pregnancy but has also been linked to other physiologic and pathologic conditions.

Macrophage migration inhibitory factor contributes to the immune escape of ovarian cancer by down-regulating NKG2D

The proinflammatory cytokine macrophage migration inhibitory factor (MIF) stimulates tumor cell proliferation, migration, and metastasis; promotes tumor angiogenesis; suppresses p53-mediated apoptosis; and inhibits antitumor immunity by largely unknown mechanisms. We here describe an overexpression of MIF in ovarian cancer that correlates with malignancy and the presence of ascites. Functionally, we find that MIF may contribute to the immune escape of ovarian carcinoma by transcriptionally down-regulating NKG2D in vitro and in vivo which impairs NK cell cytotoxicity toward tumor cells. Together with the additional tumorigenic properties of MIF, this finding provides a rationale for novel small-molecule inhibitors of MIF to be used for the treatment of MIF-secreting cancers.

Expression of toll-like receptors by human muscle cells in vitro and in vivo:TLR3 is highly expressed in inflammatory and HIV myopathies, mediates IL-8 release and up-regulation of NKG2D-ligands

The particular microenvironment of the skeletal muscle can be the site of complex immune reactions. Toll-like receptors (TLRs) mediate inflammatory stimuli from pathogens and endogenous danger signals and link the innate and adaptive immune system. We investigated innate immune responses in human muscle. Analyzing TLR1-9 mRNA in cultured myoblasts and rhabdomyosarcoma cells, we found constitutive expression of TLR3. The TLR3 ligand Poly (I:C), a synthetic analog of dsRNA, and IFN-gamma increased TLR3 levels. TLR3 was mainly localized intracellularly and regulated at the protein level. Poly (I:C) challenge 1) activated nuclear factor-kappaB (NF-kappaB), 2) increased IL-8 release, and 3) up-regulated NKG2D ligands and NK-cell-mediated lysis of muscle cells. We examined muscle biopsy specimens of 6 HIV patients with inclusion body myositis/polymyositis (IBM/PM), 7 cases of sporadic IBM and 9 nonmyopathic controls for TLR3 expression. TLR3 mRNA levels were elevated in biopsy specimens from patients with IBM and HIV-myopathies. Muscle fibers in inflammatory myopathies expressed TLR3 in close proximity of infiltrating mononuclear cells. Taken together, our study suggests an important role of TLR3 in the immunobiology of muscle, and has substantial implications for the understanding of the pathogenesis of inflammatory myopathies or therapeutic interventions like vaccinations or gene transfer.

Measurements of immune responses for establishing correlates of vaccine protection against HIV

Well-defined correlates of protective immunity are an essential component of rational vaccine development. Despite years of basic science and three HIV vaccine efficacy trials, correlates of immunological protection from HIV infection remain undefined. In December 2010, a meeting of scientists engaged in basic and translational work toward developing HIV-1 vaccines was convened. The goal of this meeting was to discuss current opportunities and optimal approaches for defining correlates of protection, both for ongoing and future HIV-1 vaccine candidates; specific efforts were made to engage young scientists. We discuss here the highlights from the meeting regarding the progress made and the way forward for a protective HIV-1 vaccine.

Heterozygous ATM mutations do not contribute to early onset of breast cancer

Ataxia telangiectasia (AT) is a recessive syndrome, including cerebellar degeneration, immunologic defects and cancer predisposition, attributed to mutations in the recently isolated ATM (ataxia telangiectasia, mutated) gene. AT is diagnosed in 1/40,000 to 1/100,000 live births, with carriers calculated to comprise approximately 1% of the population. Studies of AT families have suggested that female relatives presumed to be carriers have a 5 to 8-fold increased risk for developing breast cancer, raising the possibility that germline ATM mutations may account for approximately 5% of all breast cancer cases. The increased risk for breast cancer reported for AT family members has been most evident among younger women, leading to an age-specific relative risk model predicting that 8% of breast cancer in women under age 40 arises in AT carriers, compared with 2% of cases between 40-59 years. To test this hypothesis, we undertook a germ-line mutational analysis of the ATM gene in a population of women with early onset of breast cancer, using a protein truncation (PTT) assay to detect chain-terminating mutations, which account for 90% of mutations identified in children with AT. We detected a heterozygous ATM mutation in 2/202 (1%) controls, consistent with the frequency of AT carriers predicted from epidemiologic studies. ATM mutations were present in only 2/401 (0.5%) women with early onset of breast cancer (P = 0.6). We conclude that heterozygous ATM mutations do not confer genetic predisposition to early onset of breast cancer.

Characterization of NKR+ T-cell subsets in human bone marrow:implications for immunosurveillance of neoplasia

In addition to hematopoietic progenitors, human bone marrow contains mature T/NK lymphocytes. Valpha24Vbeta11 NKT-cells, a subset of NK receptor+ (NKR+) T-cells in humans, are rare in bone marrow, suggesting the presence of other NKR+ T-cells which may contribute to tumor surveillance. NKR+/- T-cells were examined in blood (PB), and bone marrow from donors (DM) and patients with active hematopoietic malignancy (PM), or in remission (PR). T-cells in PR & PM were enriched for CD56+ and CD57+ subsets, compared to DM. All marrow NKR+/- T-cell subsets were more activated than PB. PM and, surprisingly, PR marrow contained more activated cells than DM. CD8+ cells were significantly increased in all patient marrows and there was evidence of the formation of an effector/memory pool in malignant marrow. These data suggest that NKR+ T-cell enrichment in human bone marrow that has been exposed to neoplastic transformation is compatible with a role in localized tumor surveillance/eradication.

GM-CSF receptor targeted treatment of primary AML in SCID mice using Diphtheria toxin fused to huGM-CSF

The severe combined immunodeficient (SCID) mouse model may be used to evaluate new approaches for the treatment of acute myeloid leukemia (AML). We have previously demonstrated the killing of SCID mouse leukemia initiating cells by in vitro incubation with human GM-CSF fused to Diphtheria toxin (DT-huGM-CSF). In this report, we show that in vivo treatment with DT-huGM-CSF eliminates AML growth in SCID mice. Seven cases of AML were studied. SCID mice were treated intraperitoneally with the maximally tolerated dose of 75 microg/kg/day for 7 days. Antileukemic efficacy was determined at days 40 and 80 after transplantation, by enumerating the percentages of human cells in SCID bone marrow using flow cytometry and short tandem repeat polymerase chain reaction (STR-PCR) analysis. Four out of seven AML cases were sensitive to in vivo treatment with DT-huGM-CSF at both evaluation time points. In three of these cases, elimination of human cells was demonstrated by flow cytometry and STR-PCR. One AML case showed moderate sensitivity for DT-huGM-CSF, and growth of the two remaining AML cases was not influenced by DT-huGM-CSF. Sensitivity was correlated with GM-CSFR expression. Our data show that DT-huGM-CSF can be used in vivo to reduce growth of AML and warrant further development of DT-huGM-CSF for the treatment of human AML.

Effect of ultraviolet-B light on lymphocyte activity at doses at which normal bone marrow stem cells are preserved

Ultraviolet B (UVB) light is known to be immunosuppressive, but, probably because of a small UVC component in the emission spectra of some of the UVB lamps used, reports vary on effective dose levels. To prevent potentially lethal graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation, alloreactive donor T-cell activity must be suppressed. In this study, a narrow wavelength UVB lamp (TL01, 312 nm peak emission) was used to determine what doses of UVB were required to abolish rat lymphocyte proliferation while simultaneously preserving rat bone marrow progenitor cell and primitive hematopoietic stem cell viability. Lymphocyte proliferation, as measured by 3H-Thymidine incorporation, in response to lectin stimulation was abolished below detection at doses greater than 3,500 J/m2. When T-cell clonogenicity was measured in a limiting dilution assay, a small fraction (0.6%) was maintained at doses up to 4,000 J/m2. Cytotoxic T-lymphocyte (CTL) activity was reduced after treatment with 4,000 J/m2, but a significant level of cytotoxicity was still maintained. Natural killer cell cytolytic activity was not affected by doses up to 4,000 J/m2. At 4,000 J+m2 there was a 10% survival of colony-forming units-granulocyte-macrophage; a 1% and 4% survival of day-8 and day-12 colony-forming units-spleen, respectively; and 11% survival of marrow repopulating ability cells. Up to 25% of late cobblestone area forming cells (4 to 5 weeks), reflecting the more immature hematopoietic stem cells, were preserved in bone marrow treated with 4,000 J/m2, indicating that early stem cells are less sensitive to UVB damage than are more committed progenitor cells. Thus, a potential therapeutic window was established at approximately 4,000 J/m2 using this light source, whereby the potentially GVHD-inducing T cells were suppressed, but a sufficient proportion of the cells responsible for engraftment was maintained.