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Bristow C.L.,Cornell College | Babayeva M.A.,Cornell College | Modarresi R.,Cornell College | Mcarthur C.P.,University of Missouri - Kansas City | And 6 more authors.
Journal of Visualized Experiments | Year: 2012

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides 1. Thus, identification of cell-free correlates that directly regulate the number of CD4 + T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates. The number of stem cells that enter blood and are destined to become circulating CD4 + T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion 2. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLE CS) and the HLE CS-reactive active α 1proteinase inhibitor (α 1PI, α 1antitrypsin, SerpinA1) 3. In HIV-1 disease, α 1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active α 1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 μM, and to achieve normal levels during the symptomatic categories 4, 5. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with >220 CD4 cells/μl, CD4 counts were correlated with serum levels of active α 1PI (r 2=0.93, p<0.0001, n=26) and inactive α 1PI (r 2=0.91, p<0.0001, n=26) 5. Administration of α 1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting α 1PI participates in regulating the number of CD4 + T cells in blood 3. With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active α 1PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum α 1PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to α 1PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA 3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active α 1PI in saliva. The resulting inhibition of PPE by active α 1PI can be measured by adding the PPE substrate SA 3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/μl), the concentration of α 1PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person 6. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable 7. Thus, active α 1PI in saliva is calculated as a ratio to saliva protein content and is termed the α 1PI Index. Results presented herein demonstrate that the α 1PI Index provides an accurate and precise physiologic method for calculating CD4 counts. © 2012 Journal of Visualized Experiments.

Bristow C.L.,Institute for Human Genetics and Biochemistry | Bristow C.L.,New York Medical College | Modarresi R.,Institute for Human Genetics and Biochemistry | Modarresi R.,New York Medical College | And 16 more authors.
Discovery Medicine | Year: 2013

Dietary lipids are transported via lymph to the liver and transformed to lipoproteins which bind to members of the low density lipoprotein receptor family (LDL-RFMs). Certain LDL-RFMs, e.g., very low density lipoprotein receptor (VLDLR), are also bound by inactivated proteinase inhibitors, the most abundant being α1proteinase inhibitor (α1PI, α1antitrypsin). Inflammation/infection, including HIV-1 infection, is accompanied by low levels of CD4+ T cells and active α1PI and high levels of inactivated α1PI. By inducing LDL-RFMs-mediated cellular locomotion, active α1PI regulates the number of CD4+ T cells. We sought to investigate whether CD4+ T cells and α1PI directly impact lipoprotein levels. At the cellular level, we show that active α1PI is required for VLDLR-mediated uptake of receptor-associated cargo, specifically CD4-bound HIV-1. We show that active α1PI levels linearly correlate with LDL levels in HIV-1 infected individuals (P<0.001) and that therapeutic, weekly infusions of active α1PI elevate the number of CD4+ T cells and HDL levels while lowering LDL levels in patients on antiretroviral therapy with controlled HIV-1. Based on the unusual combination of lipodystrophy and low levels of α1PI and CD4+ T cells in HIV-1 disease, we reveal that LDL and α1PI participate in a feedback regulatory pathway. We demonstrate integral roles for sequentially acting active and inactive α1PI in the uptake and recycling of receptors and cargo aggregated with VLDLR including CD4 and chemokine receptors. Evidence supports a role for α1PI as a primary sentinel to deploy the immune system as a consequence of its role in lipoprotein transport. © Discovery Medicine.

Bristow C.L.,New York Medical College | Bristow C.L.,Institute for Human Genetics and Biochemistry | Babayeva M.A.,New York Medical College | Babayeva M.A.,Institute for Human Genetics and Biochemistry | And 3 more authors.
PLoS ONE | Year: 2012

Background: The regulation of adult stem cell migration through human hematopoietic tissue involves the chemokine CXCL12 (SDF-1) and its receptor CXCR4 (CD184). In addition, human leukocyte elastase (HLE) plays a key role. When HLE is located on the cell surface (HLE CS), it acts not as a proteinase, but as a receptor for α 1proteinase inhibitor (α 1PI, α 1antitrypsin, SerpinA1). Binding of α 1PI to HLE CS forms a motogenic complex. We previously demonstrated that α 1PI deficiency attends HIV-1 disease and that α 1PI augmentation produces increased numbers of immunocompetent circulating CD4 + lymphocytes. Herein we investigated the mechanism underlying the α 1PI deficiency that attends HIV-1 infection. Methods and Findings: Active α 1PI in HIV-1 subjects (median 17 μM, n = 35) was significantly below normal (median 36 μM, p<0.001, n = 30). In HIV-1 uninfected subjects, CD4 + lymphocytes were correlated with the combined factors α 1PI, HLE CS + lymphocytes, and CXCR4 + lymphocytes (r 2 = 0.91, p<0.001, n = 30), but not CXCL12. In contrast, in HIV-1 subjects with

Institute For Human Genetics And Biochemistry | Date: 2011-11-22

A previously unrecognized fundamental property of _(1)PI is to regulate the phenotypic composition of circulating and tissue-associated cells derived from hematopoietic stem cells. The present invention comprises screening for various unmodified and modified _(1)PIs which are useful in the treatment of abnormalities in the number of cells of myeloid or lymphoid lineage that are associated with HW-1 infection, microbial infection, leukemia, solid tumor cancers, atherosclerosis, autoimmunity, stem cell transplantation, organ transplantation, and other diseases affected by cells of the immune system. The interaction of _(1)PI with its receptors, HLE_(CS )and LRP, influences the level of cells of different lineages. Genetic and proteolytic modification of _(1)PI is used to target these receptors to increase or decrease specific cell populations, as needed, in the various disease states.

Institute For Human Genetics And Biochemistry | Date: 2012-02-03

A method for modulation of plasma membrane associated Human Leukocyte Elastase (HLE) to inflammatory states by interaction of HLE with an antagonist to inhibit HLE and thereby interruption in plasma associated events (e.g. HIV disease progression, bacterial infections and autoimmune diseases), which are responsive/sensitive to such inflammation. The antagonist suitable for use in this invention is designed to interact with each of the catalytic triad of the HLE plasma membranes protein and the lipid interactive amino acids of the HLE plasma membrane protein.

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