Role of Haptoglobin in Health and Disease: A Focus on Diabetes.

In Brief Prospective identification of individuals with diabetes who are at greatest risk for developing complications would have considerable public health importance by allowing appropriate resources to be focused on those who would benefit most from aggressive intervention. Haptoglobin (Hp) is an acute-phase protein that is crucial for the elimination of free hemoglobin and the neutralization of oxidative damage. In the past two decades, associations have been made between polymorphisms in Hp and complications arising from diabetes. Individuals with polymorphism in Hp have been shown to have significantly higher risk of developing cardiovascular disease. This review summarizes the current literature on the role of Hp in health and disease, with a focus on diabetes.

Characterization of functional mannose receptor in a continuous hybridoma cell line.

BACKGROUND: The mannose receptor is the best described member of the type I transmembrane C-type lectins; however much remains unanswered about the biology of the receptor. One difficulty has been the inability to consistently express high levels of a functional full length mannose receptor cDNA in mammalian cells. Another difficulty has been the lack of a human macrophage cell line expressing a fully functional receptor. Commonly used human macrophage cell lines such as U937, THP-1, Mono-Mac and HL60 do not express the mannose receptor. We have developed a macrophage hybridoma cell line (43MR cells) created by fusion of U937 cells with primary human monocyte-derived macrophages, resulting in a non-adherent cell line expressing several properties of primary macrophages. The purpose of this study was to identify and select mannose receptor-expressing cells using fluorescence-activated cell sorting and to characterize the expression and function of the receptor. RESULTS: In the current study we show that the mannose receptor found on this novel cell has endocytic characteristics consistent with and similar to the mannose receptor found on the surface of monocyte-derived human macrophages and rat bone marrow-derived macrophages. In addition, we demonstrate that these cells engage and internalize pathogen particles such as S. aureus and C. albicans. We further establish the transfectability of these cells via the introduction of a plasmid expressing influenza A hemagglutinin. CONCLUSIONS: The 43MR cell line represents the first naturally expressed MR-positive cell line derived from a human macrophage background. This cell line provides an important cell model for other researchers for the study of human MR biology and host-pathogen interactions.

First complete and productive cell culture model for members of the genus Iridovirus.

Chilo iridescent virus (CIV; the type strain of the genus Iridovirus) replicates productively in larvae of the boll weevil, Anthonomus grandis. This study focuses on characterizing productive infections of a boll weevil cell line, BRL-AG-3A (AG3A), starting with CIV reared in the waxworm, Galleria mellonella. We show that CIV can be continually and productively passaged to high titer in AG3A cells. The replication of larval-derived CIV in AG3A was analyzed by observing viral DNA replication and restriction endonuclease digestion profiles, morphogenesis, and infectivity using TCID(50) assays with AG3A as an indicator cell line. The data showed that virus passaged in the AG3A host is stable. AG3A cells are more efficient than previously utilized CF-124T cells from Choristoneura fumiferana. This system constitutes a superior model for cellular and molecular studies on CIV; it represents the first complete, productive cell culture model for the replication of CIV or any member of the genus Iridovirus.

Oats Lower Age-Related Systemic Chronic Inflammation (iAge) in Adults at Risk for Cardiovascular Disease.

Despite being largely preventable, cardiovascular disease (CVD) is still the leading cause of death globally. Recent studies suggest that the immune system, particularly a form of systemic chronic inflammation (SCI), is involved in the mechanisms leading to CVD; thus, targeting SCI may help prevent or delay the onset of CVD. In a recent placebo-controlled randomized clinical trial, an oat product providing 3 g of ß-Glucan improved cholesterol low-density lipoprotein (LDL) levels and lowered cardiovascular risk in adults with borderline high cholesterol. Here, we conducted a secondary measurement of the serum samples to test whether the oat product has the potential to reduce SCI and improve other clinical outcomes related to healthy aging. We investigated the effects of the oat product on a novel metric for SCI called Inflammatory Age® (iAge®), derived from the Stanford 1000 Immunomes Project. The iAge® predicts multimorbidity, frailty, immune decline, premature cardiovascular aging, and all-cause mortality on a personalized level. A beneficial effect of the oat product was observed in subjects with elevated levels of iAge® at baseline (>49.6 iAge® years) as early as two weeks post-treatment. The rice control group did not show any significant change in iAge®. Interestingly, the effects of the oat product on iAge® were largely driven by a decrease in the Eotaxin-1 protein, an aging-related chemokine, independent of a person's gender, body mass index, or chronological age. Thus, we describe a novel anti-SCI role for oats that could have a major impact on functional, preventative, and personalized medicine.

Mitogen-activated protein kinases and NFkappaB are involved in SP-A-enhanced responses of macrophages to mycobacteria.

BACKGROUND: Surfactant protein A (SP-A) is a C-type lectin involved in surfactant homeostasis as well as host defense in the lung. We have recently demonstrated that SP-A enhances the killing of bacillus Calmette-Guerin (BCG) by rat macrophages through a nitric oxide-dependent pathway. In the current study we have investigated the role of tyrosine kinases and the downstream mitogen-activated protein kinase (MAPK) family, and the transcription factor NFkappaB in mediating the enhanced signaling in response to BCG in the presence of SP-A. METHODS: Human SP-A was prepared from alveolar proteinosis fluid, and primary macrophages were obtained by maturation of cells from whole rat bone marrow. BCG-SP-A complexes were routinely prepared by incubation of a ratio of 20 microg of SP-A to 5 x 105 BCG for 30 min at 37 degrees C. Cells were incubated with PBS, SP-A, BCG, or SP-A-BCG complexes for the times indicated. BCG killing was assessed using a 3H-uracil incorporation assay. Phosphorylated protein levels, enzyme assays, and secreted mediator assays were conducted using standard immunoblot and biochemical methods as outlined. RESULTS: Involvement of tyrosine kinases was demonstrated by herbimycin A-mediated inhibition of the SP-A-enhanced nitric oxide production and BCG killing. Following infection of macrophages with BCG, the MAPK family members ERK1 and ERK2 were activated as evidence by increased tyrosine phosphorylation and enzymatic activity, and this activation was enhanced when the BCG were opsonized with SP-A. An inhibitor of upstream kinases required for ERK activation inhibited BCG- and SP-A-BCG-enhanced production of nitric oxide by approximately 35%. Macrophages isolated from transgenic mice expressing a NFkappaB-responsive luciferase gene showed increased luciferase activity following infection with BCG, and this activity was enhanced two-fold in the presence of SP-A. Finally, lactacystin, an inhibitor of IkappaB degradation, reduced BCG- and SP-A-BCG-induced nitric oxide production by 60% and 80% respectively. CONCLUSION: These results demonstrate that BCG and SP-A-BCG ingestion by macrophages is accompanied by activation of signaling pathways involving the MAP kinase pathway and NFkappaB.

Virus glycosylation: role in virulence and immune interactions.

The study of N-linked glycosylation as it relates to virus biology has become an area of intense interest in recent years due to its ability to impart various advantages to virus survival and virulence. HIV and influenza, two clear threats to human health, have been shown to rely on expression of specific oligosaccharides to evade detection by the host immune system. Additionally, other viruses such as Hendra, SARS-CoV, influenza, hepatitis and West Nile rely on N-linked glycosylation for crucial functions such as entry into host cells, proteolytic processing and protein trafficking. This review focuses on recent findings on the importance of glycosylation to viral virulence and immune evasion for several prominent human pathogens.

Precision Healthcare of Type 2 Diabetic Patients Through Implementation of Haptoglobin Genotyping.

It is well-recognized that there is a need for medicine to migrate to a platform of delivering preventative care based on an individual's genetic make-up. The US National Research Council, the National Institute of Health and the American Heart Association all support the concept of utilizing genomic information to enhance the clinical management of patients. It is believed this type of precision healthcare will revolutionize health management. This current attitude of some of the most respected institutes in healthcare sets the stage for the utilization of the haptoglobin (Hp) genotype to guide precision management in type 2 diabetics (DM). There are three main Hp genotypes: 1-1, 2-1, 2-2. The Hp genotype has been studied extensively in (DM) and from the accumulated data it is clear that Hp should be considered in all DM patients as an additional independent cardiovascular disease (CVD) risk factor. In DM patients Hp2-2 generates five times increased risk of CVD compared to Hp1-1 and three times increased risk compared to Hp2-1. Data has also shown that carrying the Hp2-2 gene in DM compared to carrying an Hp1-1 genotype can increase the risk the microvascular complications of nephropathy and retinopathy. In addition, the Hp2-2 gene enhances post percutaneous coronary intervention (PCI) complications such as, in stent restenosis and need for additional revascularization during the first-year post PCI. Studies have demonstrated significant mitigation of CVD risk in Hp2-2 DM patients with administration of vitamin E and maintaining tight glycemic control. CVD is the leading cause of death and disability in DM as well-representing a huge financial burden. As such, evaluating the Hp genotype in DM patients can enhance the predictability and management of CVD risk.

HIV-1 Nef mediates post-translational down-regulation and redistribution of the mannose receptor.

Human immunodeficiency virus (HIV) has derived a variety of means to evade the host immune response. HIV-derived proteins, including Tat, Nef, and Env, have all been reported to decrease expression of host molecules such as CD4 and major histocompatibility complex I, which would assist in limiting viral replication. The mannose receptor (MR) on the surface of macrophages and dendritic cells (DC) has been proposed to function as an effective antigen-capture molecule, as well as a receptor for entering pathogens such as Mycobacterium tuberculosis and Pneumocystis carinii. Regulation of this receptor would therefore benefit HIV in removing an additional arm of the innate immune system. Previous work has shown that MR function is reduced in alveolar macrophages from HIV-infected patients and that surface MR levels are decreased by the HIV-derived protein Nef in DC. In addition, several laboratories have shown that CD4 is removed from the surface of T cells in a manner that might be applicable to decreased MR surface expression in macrophages. In the current study, we have investigated the role of Nef in removing MR from the cell surface. We have used a human macrophage cell line stably expressing the MR as well as human epithelial cells transiently expressing CD4 and a unique CD4/MR chimeric molecule constructed from the extracellular and transmembrane domains of CD4 and the cytoplasmic tail portion of the MR. We show that the MR is reduced on the cell surface by approximately 50% in the presence of Nef and that the MR cytoplasmic tail can confer susceptibility to Nef in the CD4/MR chimera. These data suggest that the MR is a potential intracellular target of Nef and that this regulation may represent a mechanism to further cripple the host innate immune system.

Hp: an inflammatory indicator in cardiovascular disease.

Over the past decade significant advancement has occurred in the biological and pathological role that Hp has in cardiovascular disease. Hp is an acute-phase protein with a role in the neutralization and clearance of free heme. Iron has tremendous potential for initiating vascular oxidation, inflammation and exacerbating coronary atherosclerosis. Hp genotype has been linked as a prognostic biomarker of acute myocardial infarction, heart failure, restenosis and cardiac transplant rejection. The increased understanding of Hp as a biomarker has provided new insights into the mechanisms of inflammation after cardiac injury and support the concept that Hp is not only an important antioxidant in vascular inflammation and atherosclerosis, but also an enhancer of inflammation in cardiac transplant.

Stable Expression and Characterization of an Optimized Mannose Receptor.

The mannose receptor (MR) is a macrophage surface receptor that recognizes pathogen associated molecular patterns (PAMPs) from a diverse array of bacterial, fungal and viral pathogens. Functional studies of the MR are hampered by the scarcity of human cell lines that express the receptor. Current model systems available for the study of MR biology often demonstrate low levels of expression and do not retain many of the classical MR properties. Although several laboratories have reported transient and stable expression of MR from plasmids, preliminary data from our laboratory suggests that these plasmids produce a protein that lacks critical domains and is often not stable over time. In this current report we describe the generation and characterization of a novel human codon-optimized system for transient and stable MR expression. Rare codons and sequences that contribute to mRNA instability were modified to produce mRNA that is qualitatively and quantitatively improved. Confocal imaging of the transient and stably expressed optimized receptor demonstrates a distribution consistent with previous reports. To demonstrate the functional characteristics of the optimized receptor, we further show that the introduction of codon-optimized MR plasmid can confer MR-associated phagocytosis of S. aureus to non-phagocytic HeLa cells. We show that three molecules participate in the engagement and internalization of S. aureus. MR was found to colocalize with Toll-like receptor 2 (TLR2) and Rab5 following exposure to pHrodo-stained S. aureus, suggesting cooperation among the three molecules to engage and internalize the bacterial particle. This study describes a transfection capable, optimized MR receptor with functional characteristics similar to the wild type receptor and further demonstrates a new system for the continued study of MR biology and function.

Interaction of members of the heat shock protein-70 family with the macrophage mannose receptor.

The macrophage MR has been the subject of investigation for over 20 years, and several important physiological functions have been described. However, the molecular mechanisms that regulate MR signaling and trafficking during these processes still remain elusive. The focus of the current paper was to identify potential cellular MR-interacting proteins. An initial screen of binding proteins in MR-expressing cells was performed using coimmunoprecipitation, followed by identification of matching peptide sequences using proteomics and MS. The major class of binding proteins identified belonged to the heat shock family of proteins. The specific interaction of the MR with HSP70 family members was validated by Western blot analysis, ligand binding assays, and intracellular colocalization using confocal microscopy. Additional studies indicated that inhibition of the HSP BiP by treatment of cells with EGCG reduced BiP interaction with and surface expression of the MR. Studies of possible motifs within the cytoplasmic tail of the receptor suggested that a juxtamembrane dibasic sequence may contribute to the interaction with BiP. These findings suggest that the molecular association of the MR with HSP70 family members via the receptor cytoplasmic tail may contribute to MR trafficking in macrophages.

Protein glycosylation in infectious disease pathobiology and treatment.

A host of bacteria and viruses are dependent on O-linked and N-linked glycosylation to perform vital biological functions. Pathogens often have integral proteins that participate in host-cell interactions such as receptor binding and fusion with host membrane. Fusion proteins from a broad range of disparate viruses, such as paramyxovirus, HIV, ebola, and the influenza viruses share a variety of common features that are augmented by glycosylation. Each of these viruses contain multiple glycosylation sites that must be processed and modified by the host post-translational machinery to be fusogenically active. In most viruses, glycosylation plays a role in biogenesis, stability, antigenicity and infectivity. In bacteria, glycosylation events play an important role in the formation of flagellin and pili and are vitally important to adherence, attachment, infectivity and immune evasion. With the importance of glycosylation to pathogen survival, it is clear that a better understanding of the processes is needed to understand the pathogen requirement for glycosylation and to capitalize on this requirement for the development of novel therapeutics.

Chloroquine is effective against influenza A virus in vitro but not in vivo.

BACKGROUND: Chloroquine is an inexpensive and widely available 9-aminoquinolone used in the management of malaria. Recently, in vitro assays suggest that chloroquine may have utility in the treatment of several viral infections including influenza. OBJECTIVES: We sought to test whether chloroquine is effective against influenza in vivo in relevant animal models. METHODS: The effectiveness of chloroquine at preventing or ameliorating influenza following viral challenge was assessed in established mouse and ferret disease models. RESULTS: Although active against influenza viruses in vitro, chloroquine did not prevent the weight loss associated with influenza virus infection in mice after challenge with viruses expressing an H1 or H3 hemagglutinin protein. Similarly, clinical signs and viral replication in the nose of ferrets were not altered by treatment. CONCLUSIONS: Although in vitro results were promising, chloroquine was not effective as preventive therapy in vivo in standard mouse and ferret models of influenza virus infection. This dampens enthusiasm for the potential utility of the drug for humans with influenza.

N-linked glycosylation attenuates H3N2 influenza viruses.

Over the last four decades, H3N2 subtype influenza A viruses have gradually acquired additional potential sites for glycosylation within the globular head of the hemagglutinin (HA) protein. Here, we have examined the biological effect of additional glycosylation on the virulence of H3N2 influenza viruses. We created otherwise isogenic reassortant viruses by site-directed mutagenesis that contain additional potential sites for glycosylation and examined the effect on virulence in naïve BALB/c, C57BL/6, and surfactant protein D (SP-D)-deficient mice. The introduction of additional sites was consistent with the sequence of acquisition in the globular head over the past 40 years, beginning with two sites in 1968 to the seven sites found in contemporary influenza viruses circulating in 2000. Decreased morbidity and mortality, as well as lower viral lung titers, were seen in mice as the level of potential glycosylation of the viruses increased. This correlated with decreased evidence of virus-mediated lung damage and increased in vitro inhibition of hemagglutination by SP-D. SP-D-deficient animals displayed an inverse pattern of disease, such that more highly glycosylated viruses elicited disease equivalent to or exceeding that of the wild type. We conclude from these data that increased glycosylation of influenza viruses results in decreased virulence, which is at least partly mediated by SP-D-induced clearance from the lung. The continued exploration of interactions between highly glycosylated viruses and surfactant proteins may lead to an improved understanding of the biology within the lung and strategies for viral control.