Gregory B. Pott

Exercise improvement and plasma biomarker changes with intravenous treprostinil therapy for pulmonary arterial hypertension: A placebo-controlled trial

BACKGROUND Pulmonary arterial hypertension (PAH) remains a poorly understood and frequently lethal disease with few treatment options. METHODS We conducted a placebo-controlled trial of intravenous treprostinil, a prostacyclin analog, in treatment-naive PAH patients. During 12 weeks of treatment with treprostinil or placebo, we quantified 6-minute walk distance (6MW), clinical symptoms and 11 cytokines/growth factors. RESULTS Forty-two of 44 study patients had idiopathic/familial PAH in New York Heart Association (NYHA) Class III. Treprostinil increased 6MW by a placebo-corrected median of 83 meters (p = 0.008; mean increase 93 +/- 42 meters), reduced Borg score by a median 2.0 units (p = 0.02), and improved NYHA class by a median of 1.0 (p = 0.02). There was a trend toward improved survival with treprostinil (p = 0.051). Baseline plasma angiopoietin-2 (Ang-2), vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9) and platelet-derived growth factor (PDGF) were elevated compared with reported normal ranges. Treatment with treprostinil was associated with decreased Ang-2 levels. Improvement in 6MW distance after treatment was associated with reductions in Ang-2 and MMP-9 levels. Most of the cytokines and growth factors studied were not abnormal with disease nor did they change with treatment. CONCLUSIONS We conclude that treprostinil treatment significantly improved exercise capacity, dyspnea and functional class. Several plasma proteins that might track disease were abnormal at baseline, and changes were associated with improved exercise capacity.

Alpha-1-antitrypsin inhibits human immunodeficiency virus type 1

Several observations suggest the existence of potent endogenous suppressors of human immunodeficiency virus type 1 (HIV‐1) production, and inhibitors of serine proteases may participate in this effect. Alpha‐1‐antitrypsin (AAT) is the most abundant circulating serine protease inhibitor. Physiological AAT concentrations inhibited HIV‐1 production in chronically infected U1 monocytic cells, reduced virus replication in freshly infected peripheral blood mononuclear cells, and blocked infection of permissive HeLa cells. In U1 cells, AAT suppressed activation of the HIV‐1‐inducing transcription factor NF‐κB. Similar results were obtained using CE‐2072, a synthetic inhibitor of host serine proteases. HIV‐1 did not replicate in blood obtained from healthy volunteers, but marked replication was observed in blood from individuals with hereditary AAT deficiency. These results identify AAT as a candidate circulating HIV‐1 inhibitor in vivo. Two different mechanisms of AAT‐induced HIV‐1 inhibition were identified, including reduced HIV‐1 infectivity and blockade of HIV‐1 production. A novel host pathogen interaction is suggested, and an alternative strategy to treat HIV‐1‐related disease may be possible.—Shapiro, L., Pott, G. B., Ralston, A. H. Alpha‐1‐antitrypsin inhibits human immunodeficiency virus type 1. FASEB J. 15, 122–115 (2001)

α-1-Antitrypsin is an endogenous inhibitor of proinflammatory cytokine production in whole blood

Several observations suggest endogenous suppressors of inflammatory mediators are present in human blood. α‐1‐Antitrypsin (AAT) is the most abundant serine protease inhibitor in blood, and AAT possesses anti‐inflammatory activity in vitro and in vivo. Here, we show that in vitro stimulation of whole blood from persons with a genetic AAT deficiency resulted in enhanced cytokine production compared with blood from healthy subjects. Using whole blood from healthy subjects, dilution of blood with RPMI tissue‐culture medium, followed by incubation for 18 h, increased spontaneous production of IL‐8, TNF‐α, IL‐1β, and IL‐1R antagonist (IL‐1Ra) significantly, compared with undiluted blood. Dilution‐induced cytokine production suggested the presence of one or more circulating inhibitors of cytokine synthesis present in blood. Serially diluting blood with tissue‐culture medium in the presence of cytokine stimulation with heat‐killed Staphylococcus epidermidis (S. epi) resulted in 1.2‐ to 55‐fold increases in cytokine production compared with S. epi stimulation alone. Diluting blood with autologous plasma did not increase the production of IL‐8, TNF‐α, IL‐1β, or IL‐1Ra, suggesting that the endogenous, inhibitory activity of blood resided in plasma. In whole blood, diluted and stimulated with S. epi, exogenous AAT inhibited IL‐8, IL‐6, TNF‐α, and IL‐1β significantly but did not suppress induction of the anti‐inflammatory cytokines IL‐1Ra and IL‐10. These ex vivo and in vitro observations suggest that endogenous AAT in blood contributes to the suppression of proinflammatory cytokine synthesis.

Endogenous IL-32 Controls Cytokine and HIV-1 Production

IL-32, a proinflammatory cytokine that activates the p38MAPK and NF-κB pathways, induces other cytokines, for example, IL-1β, IL-6, and TNF-α. This study investigated the role of endogenous IL-32 in HIV-1 infection by reducing IL-32 with small interfering (si)RNA in freshly infected PBMC and in the latently infected U1 macrophage cell line. When PBMC were pretreated with siRNA to IL-32 (siIL-32), IL-6, IFN-γ, and TNF-α were reduced by 57, 51, and 36%, respectively, compared with scrambled siRNA. Cotransfection of NF-κB and AP-1 reporter constructs with siIL-32 decreased DNA binding of these transcription factors by 42 and 46%, respectively. Cytokine protein array analysis revealed that the inhibitory activity of siIL-32 primarily targeted Th1 and proinflammatory cytokines and chemokines, e.g., MIP-1α/β. Unexpectedly, HIV-1 production (as measured by p24) increased 4-fold in these same PBMC when endogenous IL-32 was reduced. Because IFN-γ was lower in siIL-32-treated PBMC, we blocked IFN-γ bioactivity, which enhanced the augmentation of p24 by siIL-32. Furthermore, siIL-32 reduced the natural ligands of the HIV-1 coreceptors CCR5 (MIP-1α/β and RANTES) and CXCR4 (SDF-1). Inhibition of endogenous IL-32 in U1 macrophages also increased HIV-1. When rhIL-32γ was added to these cells, p24 levels fell by 72%; however, in the same cultures IFN-α increased 4-fold. Blockade of IFN-α/β bioactivity in IL-32γ-stimulated U1 cells revealed that IFN-α conveys the anti-HIV-1 effect of rhIL-32γ. In summary, depletion of endogenous IL-32 reduced the levels of Th1 and proinflammatory cytokines but paradoxically increased p24, proposing IL-32 as a natural inhibitor of HIV-1.

Patients with Nontuberculous Mycobacterial Lung Disease Exhibit Unique Body and Immune Phenotypes

RATIONALE Among patients with nontuberculous mycobacterial lung disease is a subset of previously healthy women with a slender body morphotype, often with scoliosis and/or pectus excavatum. We hypothesize that unidentified factors predispose these individuals to pulmonary nontuberculous mycobacterial disease. OBJECTIVES To compare body morphotype, serum adipokine levels, and whole-blood cytokine responses of patients with pulmonary nontuberculous mycobacteria (pNTM) with contemporary control subjects who are well matched demographically. METHODS We enrolled 103 patients with pNTM and 101 uninfected control subjects of similar demographics. Body mass index and body fat were quantified. All patients with pNTM and a subset of control subjects were evaluated for scoliosis and pectus excavatum. Serum leptin and adiponectin were measured. Specific cytokines important to host-defense against mycobacteria were measured in whole blood before and after stimulation. MEASUREMENTS AND MAIN RESULTS Patients with pNTM and control subjects were well matched for age, gender, and race. Patients with pNTM had significantly lower body mass index and body fat and were significantly taller than control subjects. Scoliosis and pectus excavatum were significantly more prevalent in patients with pNTM. The normal relationships between the adipokines and body fat were lost in the patients with pNTM, a novel finding. IFN-γ and IL-10 levels were significantly suppressed in stimulated whole blood of patients with pNTM. CONCLUSIONS This is the first study to comprehensively compare body morphotype, adipokines, and cytokine responses between patients with NTM lung disease and demographically matched controls. Our findings suggest a novel, predisposing immunophenotype that should be mechanistically defined.

Effect of cryopreservation on measurement of cytomegalovirus-specific cellular immune responses in HIV-infected patients.

&NA;To determine the feasibility of cytomegalovirus (CMV)‐specific cellmediated immunity (CMI) studies using cryopreserved cells, we compared lymphocyte proliferation assays (LPA), responder cell frequency (RCF), interleukin‐2 (IL‐2) and interferon‐&ggr; (IFN‐&ggr;) production using fresh and cryopreserved peripheral blood mononuclear cells (PBMCs) from 53 HIV‐infected patients and 15 uninfected controls. Qualitative CMV LPA results were concordant in ≥84% of the specimens from either HIV‐infected patients or controls. Proliferation‐based RCF, IL‐2, and IFN‐&rgr; comparisons showed that cryopreservation reduces the number of CMV‐specific responders and decreases cytokine secretion, without changing the rank order of the results (p < .01). In contrast, the number of flow cytometry‐enumerated IFN‐&ggr;‐producing CD4+ cells was not significantly changed by cryopreservation. In HIV‐infected patients, the differences between fresh and frozen cell assays were not influenced by CD4 cell numbers or HIV viral load. These data indicate that cryopreserved cells are suitable for longitudinal studies of the CMV‐specific immune response in HIV‐infected patients and uninfected controls.

Immunity to Human Immunodeficiency Virus (HIV) in Children with Chronic HIV Infection Receiving Highly Active Antiretroviral Therapy

ABSTRACT Our objective was to describe the CD4-mediated human immunodeficiency virus (HIV)-specific cell-mediated immunity (CMI) and its virologic and immunologic correlates in children with chronic HIV infection on highly active antiretroviral therapy (HAART). Twelve HIV-infected children on stable antiretroviral therapy with a median level of CD4+ lymphocytes (CD4%) of 25.5% and a median viral load (VL) of 786 HIV RNA copies/ml were enrolled in this study. Nine of these children were also cytomegalovirus (CMV) seropositive. Blood mononuclear cells, stimulated with HIV and CMV antigens, were used to measure lymphocyte proliferation and to enumerate gamma interferon (IFN-γ)-producing CD4+ cells. HIV CMI and CMV CMI were detected in similar proportions of patients and correlated with each other, although the HIV responses were less robust. HIV lymphocyte proliferation significantly increased with lower HIV VL and showed a trend to increase with higher CD4% and longer time on HAART. The in vitro IFN-γ response to HIV or CMV was not affected by CD4%, VL, or HAART. Pediatric patients with established HIV infection on HAART frequently exhibit HIV CMI despite undetectable HIV replication. We concluded that the association between HIV CMI and CMV CMI indicates that the same factors govern responsiveness to either antigen.

Alpha-1-antitrypsin inhibits nitric oxide production

NO is an endogenously produced gas that regulates inflammation, vascular tone, neurotransmission, and immunity. NO production can be increased by exposing cells to several endogenous and exogenous proinflammatory mediators, including IFN‐γ, TNF‐α, IL‐1β, and LPS. As AAT has been shown to inhibit cell activation and suppress cytokine production associated with proinflammatory stimulation, we examined AAT for NO‐suppressive function. In RAW 264.7 murine macrophagic cells, physiological AAT concentrations significantly inhibited combined LPS‐ and IFN‐γ‐induced NO synthesis, and NO synthesis inhibition was associated with decreased expression of iNOS, suppressed NF‐κB activation, and reduced translocation of extracellular AAT into the interior of RAW 264.7 cells. CE‐2072, a synthetic inhibitor of serine proteases, also suppressed NO production, iNOS expression, and NF‐κB activation. However, AAT did not alter activation of intracellular MAPKs. In subjects with genetic AAT deficiency, exhaled NO was increased significantly compared with exhaled NO in healthy controls. These in vitro and in vivo studies suggest that AAT is an endogenous inhibitor of NO production. Administering AAT or AAT‐like molecules may have use as a treatment for diseases associated with excessive NO production.

Alpha-1 antitrypsin reduces severity of pseudomonas pneumonia in mice and inhibits epithelial barrier disruption and pseudomonas invasion of respiratory epithelial cells.

Nosocomial pneumonia (NP) is the third most common hospital-acquired infection and the leading cause of death due to hospital-acquired infection in the US. During pneumonia and non-pneumonia severe illness, respiratory tract secretions become enriched with the serine protease neutrophil elastase (NE). Several NE activities promote onset and severity of NP. NE in the airways causes proteolytic tissue damage, augments inflammation, may promote invasion of respiratory epithelium by bacteria, and disrupts respiratory epithelial barrier function. These NE activities culminate in enhanced bacterial replication, impaired gas exchange, fluid intrusion into the airways, and loss of bacterial containment that can result in bacteremia. Therefore, neutralizing NE activity may reduce the frequency and severity of NP. We evaluated human alpha-1 antitrypsin (AAT), the prototype endogenous NE inhibitor, as a suppressor of bacterial pneumonia and pneumonia-related pathogenesis. In AAT+/+ transgenic mice that express human AAT in lungs, mortality due to Pseudomonas aeruginosa (P.aer) pneumonia was reduced 90% compared to non-transgenic control animals. Exogenous human AAT given to non-transgenic mice also significantly reduced P.aer pneumonia mortality. P.aer-infected AAT+/+ mice demonstrated reduced lung tissue damage, decreased bacterial concentrations in lungs and blood, and diminished circulating cytokine concentrations compared to infected non-transgenic mice. In vitro, AAT suppressed P.aer internalization into respiratory epithelial cells and inhibited NE or P.aer-induced disruption of epithelial cell barrier function. The beneficial effects of human AAT in murine P.aer pneumonia raise the possibility of AAT use as a prophylactic treatment for NP in humans, and suggest a role for AAT as an innate immune mediator.

Whole-Blood Interleukin-18 Level during Early HIV-1 Infection Is Associated with Reduced CXCR4 Coreceptor Expression and Interferon-γ Levels

Interleukin (IL)-18 generates T helper 1-type immunity and inhibits human immunodeficiency virus type 1 (HIV-1) in primary cells in vitro. Because IL-18 may participate in HIV-1 containment, whole-blood IL-18 levels were measured in 20 healthy control subjects and longitudinally in 28 subjects with early HIV-1 infection. Compared with those in control subjects, IL-18 levels were higher during early HIV-1 infection, and IL-18 levels predicted reduced CXCR4 HIV-1 coreceptor expression and diminished interferon (IFN)- gamma levels. By contrast, a direct association between IL-18 and IFN- gamma levels was observed in blood stimulated with lipopolysaccharide. During early HIV-1 infection, IL-18 may regulate HIV-1 coreceptor expression and have antiretroviral activity.