Rongzhen Zhang

Circulating endotoxin and systemic immune activation in sporadic amyotrophic lateral sclerosis (sALS)

The present study reports elevated levels of endotoxin/lipopolysaccharide (LPS) concentrations in plasma from patients with sporadic amyotrophic lateral sclerosis (sALS) and Alzheimers (AD) as compared to healthy controls. Levels of plasma LPS showed a significant positive correlation with degree of blood monocyte/macrophage activation in disease groups and was most elevated in patients with advanced sALS disease. There was a significant negative relationship between plasma LPS and levels of monocyte/macrophage IL-10 expression in sALS blood. These data suggest that systemic LPS levels and activated monocyte/macrophages may play significant roles in the pathogenesis of sALS.

Systemic immune system alterations in early stages of Alzheimer's disease.

Immune activation and inflammation play significant roles in the pathogenesis of Alzheimers disease (AD). To test whether AD patients showed systemic manifestations of inflammation, blood from 41 patients with early stages of AD and 31 aged-match elderly controls were evaluated. Cellular markers for monocyte/macrophage (MO) activation and CD8 T lymphocyte were increased in early AD patients. Expression of monocyte CCR2, the receptor for monocyte chemoattractant protein-1 (MCP-1), was decreased; however, plasma MCP-1 levels were significantly increased and were related to the degree of MO activation in AD. These findings suggest that AD pathogenesis may be influenced by systemic immunologic dysfunction and provides potential immunologic targets for therapeutic intervention.

Gene expression profiling in peripheral blood mononuclear cells from patients with sporadic amyotrophic lateral sclerosis (sALS)

The aim of this study was to identify gene expression profiles in peripheral blood mononuclear cells (PBMCs) from sporadic amyotrophic lateral sclerosis (sALS) patients to gain insights into the pathogenesis of ALS. We found that upregulation of LPS/TLR4-signaling associated genes was observed in the PMBCs from sALS patients after short-term cultivation, and that elevated levels of gene expression correlated with degree of peripheral blood monocyte activation and plasma LPS levels in sALS. Similar patterns of gene expression were reproduced in LPS stimulated PBMCs from healthy controls. These data suggest that chronic monocyte/macrophage activation may be through LPS/TLR4-signaling pathways in ALS.

Increased HLA-DR expression on peripheral blood monocytes in subsets of subjects with primary HIV infection is associated with elevated CD4 T-cell apoptosis and CD4 T-cell depletion.

Whereas T-cell activation parameters of HIV disease have been extensively studied, the activation status of circulating monocytes has received less attention. Sixty-one subjects with primary HIV infection were evaluated by fluorescent-activated cell sorter (FACS) analysis at baseline (pretreatment) for CD4 T-cell count, CD4 T-cell apoptosis, and immune activation. A subset of 15 subjects with marked elevated (3 standard deviations above normal) monocyte DR expression had significantly reduced CD4 T-cell counts at baseline (p <.01) when compared with 46 subjects without monocyte activation. Ten subjects who presented with elevated levels of both CD14/DR, and CD4/CD38, had higher CD4 T-cell apoptosis (p <.001), and lower CD4 T-cell counts (p <.001) and higher baseline plasma HIV RNA (p <.01) than 21 subjects without elevated CD14/DR and CD4/CD38 coexpression. Fifty subjects were subsequently evaluated for immune cell activation over 24 weeks postinitiation of highly active antiretroviral therapy (HAART). A subgroup of 5 subjects who had persistent CD14/DR activation showed continuous depression of CD4 T-cell counts persisting for up to 2 years. The CD4 T-cell counts of this subgroup were significantly lower, at all time points, in comparison to 35 subjects who lacked any persistent expression of monocyte or CD4 T-cell activation (at 24 weeks, p <.002). We conclude that monocyte activation as defined by elevation of CD14/DR expression correlates to CD4 T-cell depletion in primary HIV infection, and is predictive of a poor CD4 T-cell response to HAART in a subset of patients.

NP001 regulation of macrophage activation markers in ALS: A phase I clinical and biomarker study

Abstract This is a phase I, placebo-controlled, single ascending dose safety and tolerability study of NP001 in patients with ALS. NP001 is a novel regulator of inflammatory macrophages and monocytes. As ALS progression is thought to be related to neuroinflammation, an additional objective of the study was to assess the effects of NP001 administration on monocyte activation markers. Thirty-two ALS patients were enrolled and received either placebo (eight) or one of four (six at each dose) ascending single i.v. doses (0.2, 0.8, 1.6 and 3.2 mg/kg NP001). Patients were monitored for safety, and blood monocyte immune activation markers CD16 and HLA-DR were assessed pre- and 24 h post-dosing. Changes from baseline were calculated. Results showed that NP001 was generally safe and well tolerated. Importantly, a single dose of NP001 caused a dose-dependent reduction in expression of monocyte CD16, a marker of monocyte activation/inflammation. Additionally, monocyte HLA-DR expression was also decreased in those patients with elevated values at baseline. In conclusion, these data indicate that NP001 has an acute effect on inflammatory monocytes in ALS patient blood. The potential for modulation of inflammation in the context of ALS disease progression will require further study with long-term follow-up.

Abstract 5026: Tumor associated targeting with Manocept: HIV associated Kaposi's sarcoma as a model system

Introduction Inflammation appears to play a critical role in tumor development of HIV-associated Kaposi9s sarcoma (KS). Specifically, emerging data show that KS tumor cells that co-express various macrophage (MO) antigens become resistant to current anti-viral therapies used to treat KS and AIDS. MOs also are a known source of KS tumor cell growth factors and substantial evidence suggests that tumor associated macrophages (TAMs) may represent a reservoir for HIV and its evolved retroviral variants. The MO pool driving these two pathological pathways share a common element rooted in the MOs, the CD206 human macrophage mannose receptor. Manocept, a molecular targeting agent, binds and enters MOs via pinocytosis of holo-CD206, providing a cell portal for the evaluation of Manocept as a MO and KS targeting agent. Methods Manocept was prepared as a synthetic molecule with a dextran backbone, and 12-20 mannose moieties. The reporter molecule (Cy3) was included as a fluorescent tracer for locating Manocept on and in MOs and KS tumor cells. Binding and localization Cy3-Manocept was assessed through intracellular visualization and flow cytometric quantitation of Cy3-Manocept binding and uptake using in vitro generation of monocyte-derived CD206+ MOs. The fresh HIV+ KS tumor tissue culture followed by immunofluorescence staining and confocal imaging was performed to confirm Cy3-Manocept uptake in KS tumor cells and TAMs. Results In vitro culture showed that Cy3-Manocept binds avidly to CD206 expressing MOs. Time course studies of increasing Cy3-Manocept concentrations confirmed continuous and ongoing uptake of Manocept into CD206+ MOs at 37°C. Fresh HIV+ KS tissue culture studies showed both Manocept uptake and CD206 staining of TAMs as well as KS tumor spindle cells (HHV8+ cells). Cy3-Manocept co-localized with CD206 in nearly all KS-associated cells expressing HHV8 and/or CD68 (Tissue MO marker), confirming that CD206 acts both as a target and Manocept concentrating receptor for TAMs and KS tumor cells. Conclusions Our results support the potential use of CD206 macrophage mannose receptor-targeted Manocept for imaging KS tumor cells, TAMs, and more importantly, for delivery of therapeutic/diagnostic agents capable of targeting all KS-associated cells including a potential MO reservoir for HIV. Citation Format: Rongzhen Zhang, Frederick O. Cope, Paige M. Bracci, Michael S. McGrath. Tumor associated targeting with Manocept: HIV associated Kaposi9s sarcoma as a model system. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5026. doi:10.1158/1538-7445.AM2015-5026

Abstract 4932: Kaposi's sarcoma represents a dynamic pathogenic process involving ongoing macrophage replenishment with both tumor cells as well as macrophages expressing CD206, a target for the recently approved imaging agent, Tilmanocept

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction In patients with Kaposis sarcoma (KS), plasma elevation of macrophage (MO)-produced growth factors such as EGF and bFGF suggests a role for tumor associated MOs (TAM) in KS pathogenesis. Goals of the current study were twofold: 1) To define MO subsets using M1 (MAC387), M2 (CD163, CD206) and pan macrophage CD68 antibodies in tissue microarray (TMA) analysis of all forms of KS including plaque, oral and visceral tumors. 2) To test whether CD206, the macrophage mannose receptor recognized by the recently approved imaging agent Tilmanocept (Navidea), is present on TAMs and KS tumor cells. If TAMs and KS tumor cells are CD206+, Tilmanocept could allow KS specific imaging for purposes of tumor staging, a procedure heretofore unapproachable. Methods A KS TMA with 66 KS cases and controls was obtained from the AIDS and Cancer Specimen Resource (ACSR). MO antigens were identified by IHC studies and results were standardized to the proportion of KSHV LANA+ cells (KS tumor specific marker). ACSR KS specimens from Kenya and South Africa (where KS is a common cause of death) were tested in a follow-up study. Results Most TAMs in KS tissues were identified with the M2 specific anti-CD163 antibody whereas the M1 anti-MAC387 antibody identified a smaller subset of cells. The CD68 antibody also identified a large number of TAMs in more than 90% of tumors. KS tumor spindle cells in general expressed macrophage antigens; however the most prevalent antigen for both KS tumor cells (LANA+) and TAMs was CD206 molecule. Expression of MO antigens and CD206 in relation to level of LANA within tumor tissues was similar across all tissue forms of KS (plaque, oral, visceral). In general, KS tissues from Africa contained higher relative levels of TAMs than tissues from US cases. Conclusions KS pathogenesis appears to involve an ongoing replenishment of TAMs from recent blood derived monocytes that express either M1 (MAC-387) or M2 (CD163) antigens. Both MO antigens identify cells that are turned over within days, suggesting that KS while appearing relatively indolent has a very dynamic MO migration component, apparently required for KS pathogenesis. This study also confirmed a preliminary study (Uccini et al. AJP 1997) that found both TAMs and KS tumor cells to express the CD206 macrophage mannose receptor. Tilmanocept, a technetium labeled ligand for CD206 recently approved by the FDA for human imaging use, may allow effective imaging of KS involved nodes and other visceral sites of disease. Considering the current inability of clinicians to determine the degree of KS spread beyond the presence of obvious skin lesions, the potential for using Tilmanocept to define tumor burden may allow earlier tumor specific treatment beyond the current use of anti-retroviral therapy alone which is proving ineffective in growing numbers of KS patients worldwide. Citation Format: Michael S. McGrath, Paige M. Bracci, Frederick O. Cope, Rongzhen Zhang. Kaposis sarcoma represents a dynamic pathogenic process involving ongoing macrophage replenishment with both tumor cells as well as macrophages expressing CD206, a target for the recently approved imaging agent, Tilmanocept. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4932. doi:10.1158/1538-7445.AM2014-4932