The Interaction between Nef Protein and ABCA1 Mutants in Tangier Disease

The genetic disorder Tangier Disease is characterized by mutations at a chromosomal locus, 9q31, which affect proper function of the cholesterol transporter ATP-Binding Cassette A1 (ABCA1). Individuals with mutant ABCA1 have very low levels of high-density lipoprotein and are at high risk for development of neuropathy and atherosclerosis. Two of the ABCA1 mutations, Q597R and R587W, lead to retention of ABCA1 in the endoplasmic reticulum (ER) in a pattern that is reminiscent of a previously reported ABCA1 inactivation by HIV-1 protein Nef. The mechanism of that inactivation involves Nef binding to an ER chaperone calnexin, which disrupts the interaction between calnexin and ABCA1 preventing proper maturation of ABCA1. As a result, ABCA1 is retained in the ER and not transported to the plasma membrane where its main activity takes place. Thus, we speculated that the underlying mechanism of retention of ABCA1 in the ER of patients with Q597R and R587W mutations is caused by a weakened interaction between mutated ABCA1 and calnexin. However, our preliminary data suggests that it is actually an abnormally strong interaction between these two molecules that leads to the retention of ABCA1 in the ER. The main aim of my research is to attempt to use HIV-1 Nef to decrease the strength of interaction between these mutants and calnexin, which may enable the transport of ABCA1 molecules to cellular membrane, thus restoring the cholesterol efflux from the affected cells. If successful, this approach could lead to a potential therapeutic treatment for Tangier disease using Nef-mimicking peptides

Alkaline phosphatase: a potential biomarker for stroke and implications for treatment

Stroke is the fifth leading cause of death in the U.S., with more than 100,000 deaths annually. There are a multitude of risks associated with stroke, including aging, cardiovascular disease, hypertension, Alzheimer’s disease (AD), and immune suppression. One of the many challenges, which has so far proven to be unsuccessful, is the identification of a cost-effective diagnostic or prognostic biomarker for stroke. Alkaline phosphatase (AP), an enzyme first discovered in the 1920s, has been evaluated as a potential biomarker in many disorders, including many of the co-morbidities associated with stroke. This review will examine the basic biology of AP, and its most common isoenzyme, tissue nonspecific alkaline phosphatase (TNAP), with a specific focus on the central nervous system. It examines the preclinical and clinical evidence which supports a potential role for AP in stroke and suggests potential mechanism(s) of action for AP isoenzymes in stroke. Lastly, the review speculates on the clinical utility of AP isoenzymes as potential blood biomarkers for stroke or as AP-targeted treatments for stroke patients

Evaluation of a novel antiviral for influenza infection in the ferret model

Influenza viruses, although common, cause thousands of deaths worldwide and remain a prominent public health issue, especially with the growing population of elderly and immune-compromised individuals. Currently, the influenza vaccine is the best tool available at protecting against infection, but the correct strains are hard to predict and the vaccines do not always work. Therefore, it is necessary to explore more effective antiviral drugs. New antivirals like these are of public health importance because they can help reduce morbidity and mortality related to influenza infection. Our collaborators from the University of Washington have computationally designed a small protein, HB36.6, which interferes with influenza infection by binding the virus’ hemagglutinin surface protein. This thesis has tested the novel antiviral using the influenza A/California/07/09 (H1N1) strain in a ferret model by looking at the effects of low and high doses of HB36.6, and comparing it to untreated controls or against the current standard antiviral, Tamiflu. Antiviral effectiveness was evaluated using a combination of clinical and viral parameters. First, the animals were monitored and scored using a detailed clinical scoring system for onset of clinical signs of disease, as well as tracked through daily measurements of weights and temperatures. Second, viral titers were quantitated using RT-PCR in tissue samples obtained at necropsy and the untreated controls were compared to HB36.6-treated animals or those treated with Tamiflu. The overall hypothesis for this study was that HB36.6 will be effective in reducing viral loads and limiting disease following aerosolized influenza infection in the ferret model, and this reduction in viral loads/disease would be more effective than the currently used antiviral, Tamiflu. The results of these studies show that HB36.6-treated animals display fewer clinical symptoms when a low dose of the treatment is used, but there appears to be no reduction in viral loads. Higher doses appear to be toxic to the animals, especially when taken over multiple days throughout the course of infection. These studies have yielded important information on a new class of influenza therapeutics that should be further evaluated using larger sample sizes

Comparison of Minnesota Teachers’ Sex Education Instructional Practices with Professional Preparation and Development

Nationally, many health teachers have not received health education training. Minnesota does not require sexual health training or sexual health professional development for health education teachers. School Health Profiles 2018 data was analyzed to determine who teaches sexual health education in Minnesota, what they teach, how they teach it, and their desired future professional development. Teachers who received pre-service health education training were compared with teachers who received other pre-service preparation. Additionally, teachers who received sexual health professional development were compared with teachers who did not receive sexual health professional development. Data analyses included crosstabs with Phi and Cramer’s V to examine any relationships between training and instructional practices. Most teachers received pre-service training in both health education and physical education. Some health education teachers in Minnesota have not received pre-service training in health education. Teachers who had pre-service training in health education were more likely to assess concepts and skills. Although most teachers want professional development on content, skills, and methods for teaching sexual health education, most have not received it in the previous two years. Professional development in sexual health was significantly associated with teaching sexual health knowledge, concepts, and skills; providing opportunities to practice skills; and assessment of concepts and skills in sexual health education. Universities need to utilize the National Sexual Health Education Teacher Preparation Standards for pre-service teacher students. Districts must hire licensed and trained health education teachers, and provide the teachers with access to sexual health education professional development

Significantly reduced CCR5-tropic HIV-1 replication in vitro in cells from subjects previously immunized with Vaccinia Virus

<p>Abstract</p> <p>Background</p> <p>At present, the relatively sudden appearance and explosive spread of HIV throughout Africa and around the world beginning in the 1950s has never been adequately explained. Theorizing that this phenomenon may be somehow related to the eradication of smallpox followed by the cessation of vaccinia immunization, we undertook a comparison of HIV-1 susceptibility in the peripheral blood mononuclear cells from subjects immunized with the vaccinia virus to those from vaccinia naive donors.</p> <p>Results</p> <p>Vaccinia immunization in the preceding 3-6 months resulted in an up to 5-fold reduction in CCR5-tropic but not in CXCR4-tropic HIV-1 replication in the cells from vaccinated subjects. The addition of autologous serum to the cell cultures resulted in enhanced R5 HIV-1 replication in the cells from unvaccinated, but not vaccinated subjects. There were no significant differences in the concentrations of MIP-1α, MIP-1β and RANTES between the cell cultures derived from vaccinated and unvaccinated subjects when measured in culture medium on days 2 and 5 following R5 HIV-1 challenge.</p> <p>Discussion</p> <p>Since primary HIV-1 infections are caused almost exclusively by the CCR5-tropic HIV-1 strains, our results suggest that prior immunization with vaccinia virus might provide an individual with some degree of protection to subsequent HIV infection and/or progression. The duration of such protection remains to be determined. A differential elaboration of MIP-1α, MIP-1β and RANTES between vaccinated and unvaccinated subjects, following infection, does not appear to be a mechanism in the noted protection.</p

Exosomes containing HIV protein Nef reorganize lipid rafts potentiating inflammatory response in bystander cells.

HIV infection has a profound effect on "bystander" cells causing metabolic co-morbidities. This may be mediated by exosomes secreted by HIV-infected cells and containing viral factors. Here we show that exosomes containing HIV-1 protein Nef (exNef) are rapidly taken up by macrophages releasing Nef into the cell interior. This caused down-regulation of ABCA1, reduction of cholesterol efflux and sharp elevation of the abundance of lipid rafts through reduced activation of small GTPase Cdc42 and decreased actin polymerization. Changes in rafts led to re-localization of TLR4 and TREM-1 to rafts, phosphorylation of ERK1/2, activation of NLRP3 inflammasome, and increased secretion of pro-inflammatory cytokines. The effects of exNef on lipid rafts and on inflammation were reversed by overexpression of a constitutively active mutant of Cdc42. Similar effects were observed in macrophages treated with exosomes produced by HIV-infected cells or isolated from plasma of HIV-infected subjects, but not with exosomes from cells and subjects infected with ΔNef-HIV or uninfected subjects. Mice injected with exNef exhibited monocytosis, reduced ABCA1 in macrophages, increased raft abundance in monocytes and augmented inflammation. Thus, Nef-containing exosomes potentiated pro-inflammatory response by inducing changes in cholesterol metabolism and reorganizing lipid rafts. These mechanisms may contribute to HIV-associated metabolic co-morbidities

Long-term changes of serum chemokine levels in vaccinated military personnel

BACKGROUND: Members of the United States Armed Forces receive a series of vaccinations during their course of service. To investigate the influence of multiple vaccinations on innate immunity, we measured concentrations of a panel of immunomodulatory and pro-inflammatory cytokines in serum samples from a group of such individuals. RESULTS: Significantly increased levels of macrophage inflammatory protein 1α (MIP-1α), MIP-1β and interleukin 8 (IL-8) were detected. Since these cytokines are known to have anti-human immunodeficiency virus (HIV) activity, we tested the effect of serum from these individuals on HIV-1 infectivity and susceptibility of their peripheral blood mononuclear cells (PBMCs) to HIV-1 infection in vitro. Sera from vaccinated military personnel inhibited, and their PBMCs were partially resistant to, infection by HIV-1 strains tropic to CCR5 (R5), but not to CXCR4 (X4), chemokine receptor. CONCLUSION: These findings demonstrate that increased anti-HIV chemokines can be detected in vaccine recipients up to 68 weeks following immunization

Targeting the Blood-Brain Barrier to Prevent Sepsis-Associated Cognitive Impairment

Sepsis is a systemic inflammatory disease resulting from an infection. This disorder affects 750 000 people annually in the United States and has a 62% rehospitalization rate. Septic symptoms range from typical flu-like symptoms (eg, headache, fever) to a multifactorial syndrome known as sepsis-associated encephalopathy (SAE). Patients with SAE exhibit an acute altered mental status and often have higher mortality and morbidity. In addition, many sepsis survivors are also burdened with long-term cognitive impairment. The mechanisms through which sepsis initiates SAE and promotes long-term cognitive impairment in septic survivors are poorly understood. Due to its unique role as an interface between the brain and the periphery, numerous studies support a regulatory role for the blood-brain barrier (BBB) in the progression of acute and chronic brain dysfunction. In this review, we discuss the current body of literature which supports the BBB as a nexus which integrates signals from the brain and the periphery in sepsis. We highlight key insights on the mechanisms that contribute to the BBB’s role in sepsis which include neuroinflammation, increased barrier permeability, immune cell infiltration, mitochondrial dysfunction, and a potential barrier role for tissue non-specific alkaline phosphatase (TNAP). Finally, we address current drug treatments (eg, antimicrobials and intravenous immunoglobulins) for sepsis and their potential outcomes on brain function. A comprehensive understanding of these mechanisms may enable clinicians to target specific aspects of BBB function as a therapeutic tool to limit long-term cognitive impairment in sepsis survivors

Systemic Inhibition of Tissue-Nonspecific Alkaline Phosphatase Alters the Brain-Immune Axis in Experimental Sepsis

Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitous enzyme present in many cells and tissues, including the central nervous system. Yet its functions at the brain-immune axis remain unclear. The goal of this study was to use a novel small molecular inhibitor of TNAP, SBI-425, to interrogate the function of TNAP in neuroimmune disorders. Following intraperitoneal (IP) administration of SBI-425, mass spectrometry analysis revealed that the SBI-425 does not cross the blood-brain barrier (BBB) in healthy mice. To elucidate the role of TNAP at the brain-immune axis, mice were subjected to experimental sepsis and received either vehicle or SBI-425 (25 mg/kg, IP) daily for 7 days. While SBI-425 administration did not affect clinical severity outcomes, we found that SBI-425 administration suppressed CD4 + Foxp3+ CD25− and CD8 + Foxp3+ CD25− splenocyte T-cell populations compared to controls. Further evaluation of SBI-425’s effects in the brain revealed that TNAP activity was suppressed in the brain parenchyma of SBI-425-treated mice compared to controls. When primary brain endothelial cells were treated with a proinflammatory stimulus the addition of SBI-425 treatment potentiated the loss of barrier function in BBB endothelial cells. To further demonstrate a protective role for TNAP at endothelial barriers within this axis, transgenic mice with a conditional overexpression of TNAP were subjected to experimental sepsis and found to have increased survival and decreased clinical severity scores compared to controls. Taken together, these results demonstrate a novel role for TNAP activity in shaping the dynamic interactions within the brain-immune axis