Reduced Susceptibility to VIRIP-Based HIV-1 Entry Inhibitors Has a High Genetic Barrier and Severe Fitness Costs.

VIRIP has been identified as natural HIV-1 inhibitor targeting the gp41 fusion peptide. An optimized analogue (VIR-576) was effective in a phase I/II clinical trial and initial studies showed that HIV-1 resistance to VIRIP-based inhibitors has a high genetic barrier. Partially resistant CXCR4 (X4)-tropic HIV-1 NL4-3 variants could be obtained, however, after more than 15 months of passaging in MT-4 cells in the presence of another derivative (VIR-353). Sequence analyses identified the accumulation of seven mutations across the HIV-1 envelope glycoprotein but outside the gp41 fusion peptide. The authors suggested that the three initial alterations conferred resistance, while subsequent changes restored viral fitness. Here, we introduced these mutations individually and in combination into X4- and CCR5 (R5)-tropic HIV-1 constructs and determined their impact on VIR-353 and VIR-576 susceptibility, viral infectivity, replication fitness, and fusogenicity. We found that essentially all seven mutations contribute to reduced susceptibility to VIRIP-based inhibitors. HIV-1 constructs containing ≥4 changes were substantially more resistant to both VIRIP-based inhibitors and the VRC34.01 antibody targeting the fusion peptide. However, they were also much less infectious and fusogenic than those harboring only the three initial alterations. Furthermore, the additional changes attenuated rather than rescued HIV-1 replication in primary human cells. Thus, the genetic barrier to HIV-1 resistance against VIRIP-based inhibitors is higher than previously suggested, and mutations reducing viral susceptibility come at a severe fitness cost that was not rescued during long-term cell culture passage.IMPORTANCE Many viral pathogens are critically dependent on fusion peptides (FPs) that are inserted into the cellular membrane for infection. Initially, it was thought that FPs cannot be targeted for therapy because they are hardly accessible. However, an optimized derivative (VIR-576) of an endogenous fragment of α1-antitrypsin, named VIRIP, targeting the gp41 FP reduced viral loads in HIV-1-infected individuals. Characterization of HIV-1 variants selected during long-term cell-culture passage in the presence of a VIRIP derivative suggested that just three mutations in the HIV-1 Env protein might be sufficient for VIRIP resistance and that four subsequent changes restored viral fitness. Here, we show that all seven mutations contribute to reduced viral susceptibility to VIRIP-based inhibitors and demonstrate that the additional changes strongly impair rather than rescue HIV-1 infectivity, fusogenicity, and replication fitness. High genetic barrier to resistance and severe fitness cost support further clinical development of this class of antiviral agents.

Therapeutics: Alpha-1 Antitrypsin Augmentation Therapy.

Subjects with alpha-1 antitrypsin deficiency who develop pulmonary disease are managed following general treatment guidelines, including disease management interventions. In addition, administration of intravenous infusions of alpha-1 proteinase inhibitor (augmentation therapy) at regular schedules is a specific therapy for individuals with AATD with pulmonary involvement.This chapter summarizes the manufacturing differences of commercially available formulations and the available evidence of the effects of augmentation therapy. Biologically, there is clear evidence of in vivo local antiprotease effects in the lung and systemic immunomodulatory effects. Clinically, there is cumulative evidence of slowing lung function decline and emphysema progression. The optimal dose of augmentation therapy is being revised as well as more individualized assessment of who needs this therapy.

α-1-Antitrypsin deficiency: clinical variability, assessment, and treatment.

The recognition of α-1-antitrypsin deficiency, its function, and its role in predisposition to the development of severe emphysema was a watershed in our understanding of the pathophysiology of the condition. This led to the concept and development of intravenous replacement therapy used worldwide to protect against lung damage induced by neutrophil elastase. Nevertheless, much remained unknown about the deficiency and its impact, although in recent years the genetic and clinical variations in manifestation have provided new insights into assessing impact, efficacy of therapy, and development of new therapeutic strategies, including gene therapy, and outcome measures, such as biomarkers and computed tomography. The current article reviews this progress over the preceding 50 years.

Imbalance between neutrophil elastase and its inhibitor α1-antitrypsin in obesity alters insulin sensitivity, inflammation, and energy expenditure.

The molecular mechanisms involved in the development of obesity and related complications remain unclear. Here, we report that obese mice and human subjects have increased activity of neutrophil elastase (NE) and decreased serum levels of the NE inhibitor α1-antitrypsin (A1AT, SerpinA1). NE null (Ela2(-/-)) mice and A1AT transgenic mice were resistant to high-fat diet (HFD)-induced body weight gain, insulin resistance, inflammation, and fatty liver. NE inhibitor GW311616A reversed insulin resistance and body weight gain in HFD-fed mice. Ela2(-/-) mice also augmented circulating high molecular weight (HMW) adiponectin levels, phosphorylation of AMP-activated protein kinase (AMPK), and fatty acid oxidation (FAO) in the liver and brown adipose tissue (BAT) and uncoupling protein (UCP1) levels in the BAT. These data suggest that the A1AT-NE system regulates AMPK signaling, FAO, and energy expenditure. The imbalance between A1AT and NE contributes to the development of obesity and related inflammation, insulin resistance, and liver steatosis.

Assessment of the clinical significance of antigenic and functional levels of α1-proteinase inhibitor (α1-Pi) in infiltrating ductal breast carcinomas.

OBJECTIVES: To determine the clinical significance of α1-proteinase inhibitor (α1-Pi) in infiltrating ductal breast carcinoma patients. DESIGN AND METHODS: Serum levels of α1-Pi, tryptic specific inhibitory capacity and α1-Pi circulating immune complexes were determined using radial immunodiffusion, BAPNA assays and ELISA, respectively. 2-DE-MS and immunohistochemistry were performed to examine α1-Pi protein expression. RESULTS: A decreased serum level of α1-Pi was found among breast cancer patients in comparison to controls. In addition, we found a significantly decreased mean level of α1-Pi in the node metastatic group when compared to node negative patients. However, the functional activity of the inhibitor did not decrease proportionately. Through 2-DE analyses, a differential expression of α1-Pi isoforms according to tumor stage and node metastatic development was found. CONCLUSIONS: Both α1-Pi levels and specific activity could be a source of complementary clinical information and may provide useful information for a better understanding of the mechanisms of metastasis.

Computational methods for studying serpin conformational change and structural plasticity.

Currently, over a hundred high-resolution structures of serpins are available, exhibiting a wide range of conformations. However, our understanding of serpin dynamics and conformational change is still limited, mainly due to challenges of monitoring structural changes and characterizing transient conformations using experimental methods. Insight can be provided, however, by employing theoretical and computational approaches. In this chapter, we present an overview of such methods, focusing on molecular dynamics and simulation. As serpin conformational dynamics span a wide range of timescales, we discuss the relative merits of each method and suggest which method is suited to specific conformational phenomena.

Comparison of the properties of rare variants of alpha1-proteinase inhibitor expressed in COS-1 cells and assessment of their potential as risk factors in human disease.

Among the more than 75 known variants of alpha(1)-proteinase inhibitor, a sub-population of rare, point mutations causing single amino acid replacements have been identified and classified as "at risk" alleles for development of pulmonary disease. In most cases, it is not clear how the amino acid replacements typical of these variants change the properties of the inhibitor to increase risk of disease in the affected individuals. To begin to address this question, we mutagenized a wild type alpha(1)-proteinase inhibitor cDNA to encode a panel of eight different point mutants reported to be associated with increased risk for development of pulmonary disease. These variants were then expressed in COS-l cells transiently transfected with plasmids containing the altered cDNAs. The effects of the mutations on the rates of secretion, cellular location, intracellular degradation, activity, stability, and tendency to aggregate were determined. Results of these studies show that, in some cases, the mutations affect the rate of secretion, the activity or both of these properties of alpha(1)-proteinase inhibitor in a manner consistent with its designation as an "at-risk" allele. In other cases, the mutations do not significantly change the properties of the inhibitor, suggesting that these may be normal variants and that their expression may not increase the risk of disease.

Assessment of bystander effect potency produced by intratumoral implantation of HSVtk-expressing cells using surrogate marker secretion to monitor tumor growth kinetics.

A molecular marker, human alpha-1-antitrypsin (hAAT) was transduced into tumor cells and its secretion was found to correlate with tumor growth or regression, allowing for an accurate and continuous measurement of tumor growth kinetics. Using this system, we investigated the therapeutic potential produced purely from the bystander effect of HSVtk+ CT26 cells to eradicate established CT26 colon carcinomas in mice by direct intratumoral implantation and subsequent ganciclovir administration. With lower ratios (0.1% and 1% of initial tumor burden), tumor growth kinetics went into a static (remission) phase of approximately 2 weeks duration before relapse and resumption of progressive tumor growth. When the number of CT26tk+ modified cells injected into the tumor equaled 10% to 100% of the initial tumor cell number the bystander effect was sufficient to completely eradicate established tumors indicating that a potent bystander killing effect is produced in this system, and that a cellular therapy based on this approach may have applications.

Tumour necrosis factor-alpha, resting energy expenditure and cachexia in cystic fibrosis.

1. We investigated the relationship between circulating tumour necrosis factor-alpha concentrations, resting energy expenditure, cachexia and altered intermediary metabolism in patients with cystic fibrosis and chronic pulmonary infection. 2. Twenty adult patients with cystic fibrosis and chronic bronchial sepsis covering a spectrum of severity of lung disease (forced expiratory volume in 1 s 30-100% of predicted) were compared with 10 age matched, healthy, non-cystic fibrosis subjects. 3. Circulating tumour necrosis factor-alpha, C-reactive protein and neutrophil elastase-alpha 1-antiproteinase complex concentrations were determined simultaneously with glycerol, non-esterified fatty acids, catecholamines, anthropometric indices and resting energy expenditure (ventilated hood method). 4. Weight, body mass index and arm muscle mass were reduced in patients with cystic fibrosis compared with healthy control subjects (P < 0.01), whereas mean resting energy expenditure was increased [121 versus 101% predicted, mean difference 19.2% (95% confidence interval 11.0-27.4%), P < 0.001]. Circulating concentrations of glycerol (P < 0.01), non-esterified fatty acids (P < 0.01), adrenaline (P < 0.05), tumour necrosis factor-alpha, C-reactive protein and neutrophil elastase-alpha 1-antiproteinase complex (P < 0.01) were increased in patients compared with control subjects [tumour necrosis factor-alpha 96.9 versus 24.7 pg/ml, mean difference 72.2 pg/ml [95% confidence interval 27.7-116.7 pg/ml), P < 0.001]. Resting energy expenditure was significantly related to tumour necrosis factor-alpha levels and forced expiratory volume in 1 s. 5. In patients with cystic fibrosis and chronic pulmonary sepsis changes in resting energy expenditure, body composition and intermediary metabolism are consistent with the systemic effects of the host inflammatory response, which may be responsible for cachexia in adult patients. In particular these changes are consistent with the action of tumour necrosis factor-alpha, which was detected in the circulation during a period of apparent clinical stability.

Pathogenesis of emphysema. Assessment of basic science concepts through clinical investigation.

It is clear that alpha 1AT deficiency leads to early onset pulmonary emphysema. With the lead provided by the deficiency state, studies aimed at the linkage between alpha 1AT and its target enzyme, neutrophil elastase, have provided useful information about the pathogenesis of emphysema due to cigarette smoking. alpha 1AT represents the predominant antielastase of the lower respiratory tract. This observation implicates neutrophil elastase as the enzyme responsible for lung destruction, since affinity studies demonstrate that alpha 1ATs physiologically relevant function is the inhibition of neutrophil elastase. However, because of the inexorably slow nature of the emphysema process, demonstration of the protease-antiprotease imbalance in the lungs of smokers has been difficult. Studies using sensitive assays for alpha 1AT function and for neutrophil elastase's presence have added new support for the protease-antiprotease theory, and evaluation of related disorders such as the adult respiratory distress syndrome and cystic fibrosis have provided corraborative evidence. Finally, studies that have indicated that the major site of the protease-antiprotease imbalance is the microenvironment of protease-producing cells offer a new direction for future research into the pathogenesis of emphysema.

Enteric protein loss measured by fecal alpha 1-antitrypsin clearance in the assessment of Crohn's disease activity: a study of children and adolescents.

We compared conventional measures of disease activity with 24-h fecal alpha 1-antitrypsin clearance in 59 children and adolescents with Crohn's disease and 30 with ulcerative colitis. Among the Crohn's patients clearance was increased significantly in those with abnormal erythrocyte sedimentation rates (ESR) (greater than 15 mm/h, p less than 0.005) and diminished serum albumin (less than 35 g/L, p less than 0.005), but not in those with an abnormal Crohn's disease activity index (CDAI less than 150). Correlation between alpha 1-antitrypsin clearance and ESR was poor (r = 0.17) as with CDAI (r = 0.09). We conclude that severity of enteric protein loss is not closely related to the degree of abnormality of commonly used measures of disease activity in young patients with Crohn's disease. In contrast, in patients with ulcerative colitis, alpha 1-antitrypsin clearance correlated more consistently with clinical assessment of inflammatory activity (p less than 0.001).