Mechanism of antithrombin III inhibition of factor VIIa/tissue factor activity on cell surfaces. Comparison with tissue factor pathway inhibitor/factor Xa-induced inhibition of factor VIIa/tissue factor activity

Recent studies have shown that antithrombin III (AT III)/heparin is capable of inhibiting the catalytic activity of factor VIIa bound either to relipidated tissue factor (TF) in suspension or to TF expressed on cell surfaces. We report studies of the mechanism of which by AT III inhibits factor VIIa bound to cell surface TF and compare this inhibitory mechanism with that of tissue factor pathway inhibitor (TFPI)-induced inhibition of factor VIIa/TF. AT III alone and AT III/heparin to a greater extent reduced factor VIIa bound to cell surface TF. Our data show that the decrease in the amount of factor VIIa associated with cell surface TF in the presence of AT III was the result of (1) accelerated dissociation of factor VIIa from cell surface TF after the binding of AT III to factor VIIa/TF complexes and (2) the inability of the resultant free factor VIIa-AT III complexes to bind effectively to a new cell surface TF site. Binding of TFPI/factor Xa to cell surface factor VIIa/TF complexes markedly decreased the dissociation of factor VIIa from the resultant quaternary complex of factor VIIa/TF/TFPI/factor Xa. Addition of high concentrations of factor VIIa could reverse the AT III-induced inhibition of cell surface factor VIIa/TF activity but not TFPI/factor Xa-induced inhibition of factor VIIa/TF activity.     

Instituting a Sugar-Sweetened Beverage Ban: Experience From a Children’s Hospital

Sugar-sweetened beverage (SSB) consumption is linked to increased weight and obesity in children and remains the major source of added sugar in the typical US diet across all age groups. In an effort to improve the nutritional offerings for patients and employees within our institution, Nationwide Children’s Hospital in Columbus, Ohio, implemented an SSB ban in 2011 in all food establishments within the hospital. In this report, we describe how the ban was implemented. We found that an institutional SSB ban altered beverage sales without revenue loss at nonvending food locations. From a process perspective, we found that successful implementation requires excellent communication and bold leadership at several levels throughout the hospital environment.Sugar-sweetened beverage (SSB) consumption is linked to increased weight and obesity in children and remains the major source of added sugar in the typical US diet across all age groups.1 For the purposes of this report, we define SSBs as soft drinks (soda or pop), fruit drinks, sports drinks, energy drinks, and any other beverages to which sugar, typically high fructose corn syrup or sucrose (table sugar), has been added.2 Although recent studies indicate an overall trending down of SSB intake, higher rates of consumption occur among low-income and minority children.1In 2010 the Ohio legislature passed the Healthy Choices for Healthy Children Act to help schools play an active role in decreasing the high childhood obesity rates.3 A component of this law includes an SSB ban in schools. In an effort to recognize the importance of and support this statewide initiative, Nationwide Children’s Hospital (NCH) partnered with leaders in the business community to gain bipartisan support for this legislation. To further emphasize support, NCH decided to lead by example and expand the hospital’s already existing Wellness Initiative to improve the nutritional offerings for patients and employees through implementation of an SSB ban in all food establishments within the hospital and approved catering companies. Such a ban would not only impact the food environment for children but also their families as well as hospital staff. In this report, we describe our implementation of an organizational SSB ban and report the beverage consumption and sales revenue for the years preceding and following the ban, as well as key steps to implementation.     

The HIV-1 proviral landscape reveals that Nef contributes to HIV-1 persistence in effector memory CD4+ T cells

Despite long-term antiretroviral therapy (ART), HIV-1 persists within a reservoir of CD4⁺ T cells that contribute to viral rebound if treatment is interrupted. Identifying the cellular populations that contribute to the HIV-1 reservoir and understanding the mechanisms of viral persistence are necessary to achieve an effective cure. In this regard, through Full-Length Individual Proviral Sequencing, we observed that the HIV-1 proviral landscape was different and changed with time on ART across naive and memory CD4⁺ T cell subsets isolated from 24 participants. We found that the proportion of genetically intact HIV-1 proviruses was higher and persisted over time in effector memory CD4⁺ T cells when compared with naive, central, and transitional memory CD4⁺ T cells. Interestingly, we found that escape mutations remained stable over time within effector memory T cells during therapy. Finally, we provided evidence that Nef plays a role in the persistence of genetically intact HIV-1. These findings posit effector memory T cells as a key component of the HIV-1 reservoir and suggest Nef as an attractive therapeutic target.     

Loss of leucine-rich repeat kinase 2 causes impairment of protein degradation pathways,accumulation of α-synuclein,and apoptotic cell death in aged mice

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson''s disease. LRRK2 is a large protein containing a small GTPase domain and a kinase domain, but its physiological role is unknown. To identify the normal function of LRRK2 in vivo, we generated two independent lines of germ-line deletion mice. The dopaminergic system of LRRK2^−/− mice appears normal, and numbers of dopaminergic neurons and levels of striatal dopamine are unchanged. However, LRRK2^−/− kidneys, which suffer the greatest loss of LRRK compared with other organs, develop striking accumulation and aggregation of α-synuclein and ubiquitinated proteins at 20 months of age. The autophagy–lysosomal pathway is also impaired in the absence of LRRK2, as indicated by accumulation of lipofuscin granules as well as altered levels of LC3-II and p62. Furthermore, loss of LRRK2 dramatically increases apoptotic cell death, inflammatory responses, and oxidative damage. Collectively, our findings show that LRRK2 plays an essential and unexpected role in the regulation of protein homeostasis during aging, and suggest that LRRK2 mutations may cause Parkinson''s disease and cell death via impairment of protein degradation pathways, leading to α-synuclein accumulation and aggregation over time.     

Identification of circulating antigen-specific CD4+ T lymphocytes with a CCR5+, cytotoxic phenotype in an HIV-1 long-term nonprogressor and in CMV infection

Antigen-specific CD4+ effector T cells primarily provide help for B-cell antibody responses and CD8+ cytotoxic T-lymphocyte (CTL) responses. We have found an expanded population of HIV-1 p24-specific, T-cell receptor V beta 17+, CD4+ T lymphocytes, defined by in vitro proliferative and interferon-gamma responses to a 15-mer Gag peptide, in the peripheral blood of an individual with long-term nonprogressive HIV-1 infection. Ex vivo, these cells were CCR5+ and CCR7-, consistent with an effector/memory function. Surprisingly, these cells highly expressed several proteins characteristic of cytotoxic lymphocytes, including TIA-1 (T-cell intracellular antigen 1; GMP-17/NKG7), granzymes A and B, CD161 (NKRP-1), and CD244 (C1.7/2B4). Following in vitro peptide stimulation, these cells produced interleukin 2 (IL-2) and intracellular CD40L, suggesting possible helper function, in addition to induction of perforin and cytotoxicity. A subset of cytomegalovirus (CMV)-specific CD4+ T cells in healthy adults similarly expressed these CTL markers and CCR5, ex vivo. Furthermore, this distinct subset of CD4+ T cells was significantly elevated in healthy CMV-seropositive adults, compared with CMV-seronegative individuals. These results suggest that CCR5+ CD4+ CTL may be a major effector mechanism of the immune response to viral infections in humans. Moreover, expression of CCR5 may render them particularly susceptible to cytopathic effects during progressive HIV-1 infection.     

The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases

The vast majority of eukaryotes (fungi, plants, animals, slime mold, and euglena) synthesize Asn-linked glycans (Alg) by means of a lipid-linked precursor dolichol-PP-GlcNAc2Man9Glc3. Knowledge of this pathway is important because defects in the glycosyltransferases (Alg1-Alg12 and others not yet identified), which make dolichol-PP-glycans, lead to numerous congenital disorders of glycosylation. Here we used bioinformatic and experimental methods to characterize Alg glycosyltransferases and dolichol-PP-glycans of diverse protists, including many human pathogens, with the following major conclusions. First, it is demonstrated that common ancestry is a useful method of predicting the Alg glycosyltransferase inventory of each eukaryote. Second, in the vast majority of cases, this inventory accurately predicts the dolichol-PP-glycans observed. Third, Alg glycosyltransferases are missing in sets from each organism (e.g., all of the glycosyltransferases that add glucose and mannose are absent from Giardia and Plasmodium). Fourth, dolichol-PP-GlcNAc2Man5 (present in Entamoeba and Trichomonas) and dolichol-PP- and N-linked GlcNAc2 (present in Giardia) have not been identified previously in wild-type organisms. Finally, the present diversity of protist and fungal dolichol-PP-linked glycans appears to result from secondary loss of glycosyltransferases from a common ancestor that contained the complete set of Alg glycosyltransferases.     

Early proliferation of CCR5(+) CD38(+++) antigen-specific CD4(+) Th1 effector cells during primary HIV-1 infection

We investigated whether HIV-1 antigen-specific CD4(+) T cells expressed the viral coreceptor CCR5 during primary HIV-1 infection (PHI). In the peripheral blood of subjects with very early PHI (< 22 days after onset of symptoms), there was a 10- to 20-fold increase in the proportion of highly activated (CD38(+++)) and proliferating (Ki-67(+)) CD4(+) T cells that expressed CCR5(+), and were mostly T-cell intracellular antigen-1 (TIA-1)(+) perforin(+) granzyme B(+). Inthe same patient samples, CD4(+) T cells producing interferon (IFN)-gamma in response to HIV group-specific antigen (Gag) peptides were readily detected (median, 0.58%) by intracellular cytokine assay-these cells were again predominantly CD38(+++), Ki-67(+), and TIA-(++), as well as Bcl-2(low). On average, 20% of the Gag-specific CD4(+) T cells also expressed interleukin-2 (IL-2) and were CD127 (IL-7R)(+). Taken together, these results suggest that Gag-specific T-helper 1 (Th1) effector cells express CCR5 during the primary response and may include precursors of long-term self-renewing memory cells. However, in PHI subjects with later presentation, antigen-specific CD4(+) T cells could not be readily detected (median, 0.08%), coinciding with a 5-fold lower level of the CCR5(+)CD38(+++) CD4(+) T cells. These results suggest that the antiviral response to HIV-1 infection includes highly activated CCR5(+)CD4(+) cytotoxic effector cells, which are susceptible to both apoptosis and cytopathic infection with HIV-1, and rapidly decline.     

Natural killer-like T-cell lymphomas: aggressive lymphomas of T-large granular lymphocytes [see comments]

Natural killer (NK)-like T cells are major histocompatibility complex- unrestricted cytotoxic T cells that are surface CD3-positive, express NK-cell antigens, and rearrange their T-cell receptor. Most neoplasms arising from this T-cell subpopulation have been a chronic lymphoproliferative disease referred to as T-large granular lymphocyte (LGL) leukemia. Only 10 NK-like T-cell lymphomas have been described in detail previously; this study presents the clinicopathologic features of six others and distinguishes these lymphomas from T-LGL leukemia. All patients presented with B-symptoms and often had marked hepatosplenomegaly without significant peripheral lymphadenopathy. Four of the six patients were immunosuppressed. All had CD3, CD8, CD56- positive tumors, presumably of hepatosplenic (n = 3), intestinal (n = 1), pulmonary (n = 1), or nodal (n = 1) origin. Three patients had lymphomatous bone marrow infiltrates, and four had peripheral blood involvement by neoplastic large lymphocytes, some of which had a blastic appearance or resembled virocytes. Azurophilic granules, ultrastructurally corresponding to cytoplasmic dense core and/or double density granules, were seen in all cases. T-cell clonality was shown in five tumors by Southern blot analysis, and three had abnormal karyotypes. Two untreated patients died 20 days after presentation, and three patients who received combination chemotherapy died within 5 months of presentation. One patient remains in complete remission 22 months after treatment. These findings suggest NK-like T-cell lymphomas are aggressive, are clinicopathologically distinct from T-LGL leukemia, and should be in the differential diagnosis of extranodal T-cell lymphoproliferations, including those in immunosuppressed patients. Furthermore, the LGL morphology, phenotype, and tissue distribution of some NK-like T-cell lymphomas suggest they arise from thymic- independent T cells of the hepatic sinusoids and intestinal mucosa.