Jeffrey Laurence

HIV Envelope gp120-mediated Regulation of Osteoclastogenesis via Receptor Activator of Nuclear Factor κB Ligand (RANKL) Secretion and Its Modulation by Certain HIV Protease Inhibitors through Interferon-γ/RANKL Cross-talk

Accelerated bone resorption leading to osteopenia and osteoporosis has been noted in human immunodeficiency virus (HIV) seropositive, treatment-naive patients, but it may be greatly increased in incidence in those receiving highly active anti-retroviral therapies that incorporate certain protease inhibitors (PI). The pathophysiology of these processes is unclear. We have documented the induction of the primary cytokine responsible for osteoclast differentiation and bone resorption, the receptor activator of nuclear factor κB ligand (RANKL), in T cells exposed to soluble HIV-1 envelope glycoprotein gp120. Using a murine osteoclast precursor cell line as well as primary human osteoclast precursors, we demonstrate that pharmacologic levels of two PIs that are linked clinically to osteopenia, ritonavir and saquinavir, abrogate a physiological block to RANKL activity, interferon-γ-mediated degradation of the RANKL signaling adapter protein, TRAF6 (tumor necrosis factor receptor-associated protein 6) in proteasomes. In contrast, indinavir and nelfinavir, PIs that may promote or stabilize bone formation in vivo, had no impact on this system. These findings offer a molecular basis for the acceleration of bone resorption by certain PIs and provide the first example of clinically useful drugs that can interfere with the cross-talk between RANKL and interferon-γ via the proteasome. They also suggest a novel therapeutic approach to HIV osteopenia through modulation of these two molecules.

HIV-1 upregulates Fas ligand expression in CD4+ T cells in vitro and in vivo: association with Fas-mediated apoptosis and modulation by aurintricarboxylic acid.

CD4+ T‐lymphocyte apoptosis has been associated with human immunodeficiency virus (HIV)‐1 infection in vitro, paralleling the expression of Fas (APO‐1, CD95) on peripheral blood mononuclear cells from patients with HIV disease. However, the link between Fas induction, T‐cell activation, and cell death is unclear. We document, for the first time, marked upregulation of expression of mRNA for the ligand for Fas in peripheral blood mononuclear cells from HIV seropositive individuals, and demonstrate the ability of HIV infection to induce such expression in CD4+ T cells in vitro. We also define the relevance of this expression to HIV‐mediated CD4+ T cell death. Our ability to downregulate Fas ligand message and suppress HIV‐mediated apoptosis with aurintricarboxylic acid, a clinically used protease inhibitor with known activity against programmed cell death in other systems, may open up a new area of therapy for HIV infection.

HIV-1 Vpr enhances production of receptor of activated NF-κB ligand (RANKL) via potentiation of glucocorticoid receptor activity

Summary.The HIV-1 accessory protein Vpr potentiates glucocorticoid (GC)-induced inhibition of a variety of immunologically important cytokines. We report the first instance of synergy between Vpr and GC in induction of a T cell cytokine, one which may underlie a metabolic complication of HIV infection. Accelerated bone resorption is an important complication of HIV disease and its treatment. Receptor of activated NF-κB ligand (RANKL) is the final effector of osteoclast differentiation and bone resorption. It is induced by exogenous GC, a prominent cofactor in bone mineral loss, as well as by elevated levels of endogenous GC, found in many patients with HIV disease. We document Vpr-mediated upregulation of RANKL, the dependence of this effect on GC receptor integrity, its function through a classic GC receptor motif, and its independence from Vpr-mediated G2 cell cycle arrest. These data suggest a positive regulatory role for Vpr in transcriptional control of a cytokine that may be critical to one metabolic complication of HIV.

Eculizumab in the treatment of refractory idiopathic thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is an acute thrombotic microangiopathy with a high morbidity and mortality if untreated. Refractory TTP represents a unique clinical challenge, and few therapies have proven long-term efficacy in these cases. A related thrombotic microangiopathy, atypical haemolytic uraemic syndrome (aHUS), has been successfully treated with the anti-C5 monoclonal antibody eculizumab in both the refractory setting and in patients requiring chronic plasma exchange (Waters & Licht, 2011). However, complement-based therapy has not been explored in TTP, despite many of its shared pathological features with aHUS. We report a 27-year-old white male with severe idiopathic TTP characterized by thrombocytopenia, microangiopathic haemolytic anaemia, renal failure, seizures, and coma. Stool cultures for Shiga toxin-producing Escherichia coli were negative. Plasma ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity was <5% and the ADAMTS13 IgG inhibitor titre was elevated at 6·4 units (normal <0·4). Analysis of complement and complement regulatory proteins did not reveal abnormalities in protein levels of Factor H and Factor B, and mutational analysis revealed normal alleles in CD46 and Factor I. Daily plasma exchanges produced no improvement in clinical symptoms or laboratory values and the patient’s clinical condition continued to deteriorate (Fig 1A). The plasma lactate dehydrogenase (LDH) rose, from 476 u/l on presentation to 2230 u/l by day 11. Vincristine, rituximab, steroids, and N-acetylcysteine, utilized because of its effect on high molecular weight von Willebrand factor (VWF) multimers (Chen et al, 2011), were all ineffective over several weeks. A skin biopsy performed on day 20 revealed endothelial deposition of C3d, C4d, and C5b-9, the terminal membrane attack complex (MAC) (Fig 1B). Because of the heavy complement deposition seen in the perivasculature and the patient’s critical illness, unresponsive to multiple modalities, emergency institutional review board approval was obtained to administer eculizumab. Platelet counts increased following eculizumab administration by day 29, and creatinine and LDH decreased to normal levels (Fig 1A). ADAMTS13 activity normalized to 85% by day 54. Plasma exchange and eculizumab were discontinued, and steroids were tapered starting at hospital discharge (day 57). Four weeks later, his platelet count and haemoglobin dropped rapidly, creatinine rose, and ADAMTS13 activity fell to 8·6%. Reinstituting eculizumab alone restored normal platelet, haemoglobin, and creatinine levels within 9 d, and this response was sustained for 6 weeks on bi-weekly eculizumab infusions. Two further disease flares occurred, manifested by declines in platelet counts and rises in creatinine levels, each time reversed by eculizumab. Complement dysregulation is well characterized in aHUS. Excessive MAC and complement C5a formation that is triggered by inflammation in aHUS can initiate endothelial damage, platelet activation, and secretion of ultra-large molecular weight VWF multimers (Roumenina et al, 2011). In the setting of impaired complement regulation, endothelial cell damage and thrombosis continues unabated. Mutations in complement regulatory proteins Factor I, CD46, and Factor H along with complement C3 and Factor B account for disease susceptibility in up to 60% of cases (Sanchez-Corral & Melgosa, 2010; Roumenina et al, 2011; Waters & Licht, 2011). Antibodies against Factor H have also been described in paediatric cases of aHUS, suggesting that acquired defects in alternative pathway regulation participate in thrombotic microangiopathies (Mache et al, 2009). Complement or complement regulatory factor abnormalities have not been identified in all cases of aHUS, however, and do not predict clinical response to eculizumab treatment (Mache et al, 2009; Waters & Licht, 2011). Complement regulatory factor mutations have not been systematically examined in TTP patients. In this patient with idiopathic TTP, analysis of four of such factors, those most important in aHUS, did not reveal abnormalities, although other complement and complement regulatory factor abnormalities could have been present. Multiple lines of evidence for complement participation in TTP pathology do exist, however. For example, a Factor H mutation was described in one patient with TTP and a familial ADAMTS13 deficiency; this patient had a clinical presentation distinct from than her sister, who suffered only the ADAMTS13 mutation (Noris et al, 2005). In addition, endothelial cell injury can be recapitulated in vitro by exposing microvascular endothelial cells to TTP plasma or serum, which results in MAC deposition, a process that is reversible by complement blockade (Mitra et al, 1997; Ruiz-Torres et al, 2005). Generation of MAC and C5a leads to endothelial cell damage in aHUS, resulting in endothelial apoptosis that leads to complement activation, platelet activation, and release of VWF (Dragon-Durey & Fremeaux-Bacchi, 2005; Raife, 2005). These downstream effects further enhance thrombosis and up-regulate the complement cascade, creating a positive feedback system, whereby endothelial cell damage and apoptosis further Correspondence

Low Bone Mass and High Bone Turnover in Postmenopausal Human Immunodeficiency Virus-Infected Women

CONTEXT Low bone mineral density (BMD) is commonly reported in young men and women with HIV infection, and fracture rates may be higher. With effective antiretroviral therapy (ART), the HIV population is aging. However, little is known about the skeletal status of postmenopausal women. OBJECTIVE We aimed to assess the effects of HIV infection and ART on BMD and bone turnover in postmenopausal minority women. DESIGN, SETTING, AND PATIENTS A prospective cohort study was performed in 92 HIV+ and 95 HIV- postmenopausal Hispanic and African-American women. MAIN OUTCOME MEASURES We measured BMD by dual-energy x-ray absorptiometry, fracture prevalence, serum levels of inflammatory cytokines (TNFalpha, IL-6), bone turnover markers, calciotropic hormones, and estrone. RESULTS HIV+ women were younger (56 +/- 1 vs. 60 +/- 1 yr; P < 0.01) and had lower BMI (28 +/- 1 vs. 30 +/- 1 kg/m(2); P < 0.01) and estrone levels. Prevalence of T scores below -1.0 was greater in HIV+ women at the spine (78 vs. 64%; P < 0.05), total hip (45 vs. 29%; P < 0.05), and femoral neck (64 vs. 46%; P < 0.05), and Z scores adjusted for BMI were lower in HIV+ women at the same sites. Serum TNFalpha, N-telopeptide, and C-telopeptide were significantly higher in HIV+ than HIV- women, particularly those receiving ART. HIV+ status was independently and negatively associated with spine and hip BMD after adjustment for age, ethnicity, BMI, and alcohol. CONCLUSION The lower BMD, higher prevalence of low BMD, and higher levels of bone turnover markers detected in HIV+ postmenopausal minority women could place them at high risk for future fractures.

Acquired immunodeficiency without evidence of infection with human immunodeficiency virus types 1 and 2

There have been three published cases of acquired immunodeficiency in which no evidence for infection with human immunodeficiency virus (HIV) types 1 and 2 was found. We have identified five other individuals, from the New York City area (four who have known risk factors for HIV infection), with profound CD4 depletion and clinical syndromes consistent with definitions of the acquired immunodeficiency syndrome (AIDS) or AIDS-related complex. None had evidence of HIV-1, 2 infection, as judged by multiple serologies over several years, standard viral co-cultures for HIV p24 Gag antigen, and proviral DNA amplification by polymerase chain reaction.

Role of caspases 1 and 3 and Bcl‐2‐related molecules in endothelial cell apoptosis associated with thrombotic microangiopathies

We have defined an in vitro model for the study of microvascular endothelial cell (EC) apoptosis mediated by plasma from patients with various forms of thrombotic thrombocytopenic purpura (TTP) and hemolytic‐uremic syndrome (HUS). This system reproduces a variety of histopathologic and ultrastructural features of tissue EC involved in TTP/sporadic HUS, suggesting that apoptotic EC injury is a primary pathophysiologic event in the thrombotic microangiopathies. We now document the ability of tetrapeptide‐based inhibitors of interleukin 1β‐converting enzyme (ICE)‐like caspase 1 and cysteine protease protein (CPP)‐32‐like caspase 3, two members of a novel class of cysteine proteases involved in final pathways to apoptosis, to block TTP/sporadic HUS plasma‐mediated apoptosis. Overexpression of Bcl‐XL via gene transfer suppressed this apoptosis by 70%. Transduction of EC with the Bcl‐2 homolog A1 had a more limited protective effect. These findings support a role for apoptosis‐linked cysteine proteases in the pathophysiology of TTP and sporadic HUS, and raise the possibility that specific apoptosis inhibitors may have a role in the experimental therapeutics of these syndromes. Am. J. Hematol. 59:279–287, 1998.

Noncanonical Wnt signaling promotes osteoclast differentiation and is facilitated by the human immunodeficiency virus protease inhibitor ritonavir

Wnt proteins that signal via the canonical Wnt/β-catenin pathway directly regulate osteoblast differentiation. In contrast, most studies of Wnt-related effects on osteoclasts involve indirect changes. While investigating bone mineral density loss in the setting of human immunodeficiency virus (HIV) infection and its treatment with the protease inhibitor ritonavir (RTV), we observed that RTV decreased nuclear localization of β-catenin, critical to canonical Wnt signaling, in primary human and murine osteoclast precursors. This occurred in parallel with upregulation of Wnt5a and Wnt5b transcripts. These Wnts typically stimulate noncanonical Wnt signaling, and this can antagonize the canonical Wnt pathway in many cell types, dependent upon Wnt receptor usage. We now document RTV-mediated upregulation of Wnt5a/b protein in osteoclast precursors. Recombinant Wnt5b and retrovirus-mediated expression of Wnt5a enhanced osteoclast differentiation from human and murine monocytic precursors, processes facilitated by RTV. In contrast, canonical Wnt signaling mediated by Wnt3a suppressed osteoclastogenesis. Both RTV and Wnt5b inhibited canonical, β-catenin/T cell factor-based Wnt reporter activation in osteoclast precursors. RTV- and Wnt5-induced osteoclast differentiation were dependent upon the receptor-like tyrosine kinase Ryk, suggesting that Ryk may act as a Wnt5a/b receptor in this context. This is the first demonstration of a direct role for Wnt signaling pathways and Ryk in regulation of osteoclast differentiation, and its modulation by a clinically important drug, ritonavir. These studies also reveal a potential role for noncanonical Wnt5a/b signaling in acceleration of bone mineral density loss in HIV-infected individuals, and illuminate a potential means of influencing such processes in disease states that involve enhanced osteoclast activity.

WNT/β-Catenin Signaling Is Involved in Regulation of Osteoclast Differentiation by Human Immunodeficiency Virus Protease Inhibitor Ritonavir : Relationship to Human Immunodeficiency Virus-Linked Bone Mineral Loss

Untreated human immunodeficiency virus (HIV) infection is accompanied by reduced bone mineral density, which appears to be exacerbated by certain HIV protease inhibitors (PIs). The mechanisms leading to this apparent paradox, however, remain unclear. We have previously shown that, the HIV envelope glycoprotein gp120 used at levels similar those in plasmas of untreated HIV(+) patients, induced expression of the osteoclast (OC) differentiation factor RANKL in CD4+ T cells. In addition, the HIV PI ritonavir abrogated the interferon-gamma-mediated degradation of the RANKL nuclear adapter protein TRAF6, a physiological block to RANKL activity. Here, using oligonucleotide microarrays and quantitative polymerase chain reaction, we explored potential upstream mechanisms for these effects. Ritonavir, but not the HIV PIs indinavir or nelfinavir, up-regulated the production of transcripts for OC growth factors and the non-canonical Wnt Proteins 5B and 7B as well as activated promoters of nuclear factor-kappaB signaling, but suppressed genes involved in canonical Wnt signaling. Similarly, ritonavir blocked the cytoplasmic to nuclear translocation of beta-catenin, the molecular node of the Wnt signaling pathway, in association with enhanced beta-catenin ubiquitination. Exposure of OC precursors to LiCl, an inhibitor of the canonical Wnt antagonist GSK-3beta, suppressed OC differentiation, as did adenovirus-mediated overexpression of beta-catenin. These data identify, for the first time, a biologically relevant role for Wnt signaling via beta-catenin in isolated OC precursors and the modulation of Wnt signaling by ritonavir. The reversal of these ritonavir-mediated changes by interferon-gamma provides a model for possible intervention in this metabolic complication of HIV therapy.

Thienopyridine‐linked thrombotic microangiopathy: association with endothelial cell apoptosis and activation of MAP kinase signalling cascades

The thienopyridine platelet antagonist ticlopidine is associated with development of thrombotic thrombocytopenic purpura (TTP) but the pathophysiology of this link is unclear. Severe deficiency of disintegrin and metalloproteinase with thrombospondin motif‐13 (ADAMTS13), described in familial cases and a significant fraction of idiopathic TTP, has been reported in only a few ticlopidine‐linked cases. As ticlopidine can disrupt production of extracellular matrix (ECM) components critical to microvascular endothelial cell (MVEC) integrity in vitro, we explored the hypotheses that ticlopidine and ticlopidine‐linked TTP plasmas induce MVEC apoptosis in a manner similar to that of idiopathic TTP plasmas, and that ECM components and related mitogen‐activated protein kinase (MAPK) signalling cascades may be involved in this process. Replicating the activity of plasmas from patients with idiopathic TTP, plasma from five ticlopidine‐linked TTP patients induced apoptosis of primary human dermal, glomerular and hepatic MVEC, but had no effect on pulmonary MVEC or large vessel endothelial cells (EC). Pharmacological levels of ticlopidine initiated apoptosis with similar EC lineage restriction. In parallel, ticlopidine and plasmas from idiopathic and ticlopidine‐TTP patients decreased transcripts for the ECM component thrombospondin‐1 in MVEC, but not in large vessel EC. These changes were accompanied by prolonged induction of MAPKs extracellular signal‐related kinase (ERK)‐1/2 and p38 only in TTP susceptible MVEC. Induction of apoptosis by ticlopidine and TTP plasma was abrogated by inhibitors of ERK‐1/2 and p38 phosphorylation. In conclusion, MVEC apoptosis related to altered ECM–MVEC interactions may be a key part of the pathology of ticlopidine‐linked and idiopathic TTP.