Mucociliary dysfunction in HIV and smoked substance abuse

Impaired mucociliary clearance (MCC) is a hallmark of acquired chronic airway diseases like chronic bronchitis associated with chronic obstructive pulmonary disease (COPD) and asthma. This manifests as microbial colonization of the lung consequently leading to recurrent respiratory infections. People living with HIV demonstrate increased incidence of these chronic airway diseases. Bacterial pneumonia continues to be an important comorbidity in people living with HIV even though anti-retroviral therapy has succeeded in restoring CD4+ cell counts. People living with HIV demonstrate increased microbial colonization of the lower airways. The microbial flora is similar to that observed in diseases like cystic fibrosis and COPD suggesting that mucociliary dysfunction could be a contributing factor to the increased incidence of chronic airway diseases in people living with HIV. The three principal components of the MCC apparatus are, a mucus layer, ciliary beating, and a periciliary airway surface liquid (ASL) layer that facilitates ciliary beating. Cystic fibrosis transmembrane conductance regulator (CFTR) plays a pivotal role in regulating the periciliary ASL. HIV proteins can suppress all the components of the MCC apparatus by increasing mucus secretion and suppressing CFTR function. This can decrease ASL height leading to suppressed ciliary beating. The effects of HIV on MCC are exacerbated when combined with other aggravating factors like smoking or inhaled substance abuse, which by themselves can suppress one or more components of the MCC system. This review discusses the pathophysiological mechanisms that lead to MCC suppression in people living with HIV who also smoke tobacco or abuse illicit drugs

A dual function TAR Decoy serves as an anti-HIV siRNA delivery vehicle

The TAR RNA of HIV was engineered as an siRNA delivery vehicle to develop a combinatorial therapeutic approach. The TAR backbone was found to be a versatile backbone for expressing siRNAs. Upon expression in human cells, pronounced and specific inhibition of reporter gene expression was observed with TARmiR. The resulting TARmiR construct retained its ability to bind Tat and mediate RNAi. TARmiR was able to inhibit HIV gene expression as a TAR decoy and by RNA interference when challenged with infectious proviral DNA. The implications of this dual function therapeutic would be discussed

Albuterol Modulates Its Own Transepithelial Flux via Changes in Paracellular Permeability

Although inhaled bronchodilators are commonly used in the treatment of airway disease to dilate airway smooth muscle, little is known regarding the mechanisms that regulate albuterol movement across the epithelium to reach its target, the airway smooth muscle. Because the rate of onset depends on the transepithelial transport of albuterol, to determine the mechanisms that regulate the transepithelial movement of albuterol is essential. Human bronchial epithelial cells, fully redifferentiated in culture at the air–liquid interface, were used to study the cellular uptake and total transepithelial flux of 3 H-albuterol from the apical to the basolateral surfaces. 3 H-mannitol and transepithelial electrical resistance were used to quantify changes in paracellular permeability. The majority of albuterol flux across the epithelium occurred via the paracellular route. The cellular uptake of albuterol was found to be saturable, whereas transepithelial flux was not. Cellular uptake could be inhibited by the amino acids lysine and histidine, with no effect on net transepithelial flux. Transepithelial flux was altered by maneuvers that collapsed or disrupted intercellular junctions. Acidification, usually seen in exacerbations of airway disease, decreased albuterol flux. In addition, albuterol increased its own paracellular permeability. The ability of albuterol to modulate paracellular permeability was blocked by the β 2 -adrenergic receptor–selective antagonist ICI 118551. Albuterol mainly crosses the epithelium via the paracellular pathway, but has the ability to modulate its own permeability through changes in the leakiness of tight junctions, which is modulated through the signaling of the β 2 -adrenergic receptor

A conditional RNA Pol II mono-promoter drives HIV-inducible, CRISPR-mediated cyclin T1 suppression and HIV inhibition

Gene editing using clustered regularly interspaced short palindromic repeats (CRISPR) targeted to HIV proviral DNA has shown excision of HIV from infected cells. However, CRISPR-based HIV excision is vulnerable to viral escape. Targeting cellular co-factors provides an attractive yet risky alternative to render viral escape irrelevant. Cyclin T1 is a critical modulator of HIV transcription and mediates recruitment of positive transcription elongation factor-b (P-TEFb) kinase for transcriptional elongation. Hence, a CRISPR-mediated cyclin T1 inactivation will silence HIV transcription, locking it in an inactive form in the cell and thereby serving as an effective antiviral and possibly effecting a functional cure. However, cellular genes play important roles, and their uncontrolled inhibition can promote undesirable effects. Here, we demonstrate a conditional inducible RNA polymerase II (RNA Pol II) mono-promoter-based co-expression of a CRISPR system targeting cyclin T1 from a single transcription unit. Co-expression of guide RNA (gRNA) and CRISPR-associated protein (Cas9) is observed only in HIV-infected cells and leads to sustained HIV suppression in stringent chronically infected cell lines as well as in T cell lines. We further show that incorporation of cis-acting ribozymes immediately upstream of the gRNA further enhances HIV silencing

Transforming Growth Factor-β1 and Cigarette Smoke Inhibit the Ability of β 2

Chronic bronchitis, caused by cigarette smoke exposure, is characterized by mucus hypersecretion and reduced mucociliary clearance (MCC). Effective MCC depends, in part, on adequate airway surface liquid. Cystic fibrosis transmembrane conductance regulator (CFTR) provides the necessary osmotic gradient for serosal to mucosal fluid transport through its ability to both secrete Cl(−) and regulate paracellular permeability, but CFTR activity is attenuated in chronic bronchitis and in smokers. β(2)-adrenergic receptor (β(2)-AR) agonists are widely used for managing chronic obstructive pulmonary disease, and can activate CFTR, stimulate ciliary beat frequency, and increase epithelial permeability, thereby stimulating MCC. Patients with chronic airway diseases and cigarette smokers demonstrate increased transforming growth factor (TGF)-β1 signaling, which suppresses β(2)-agonist–mediated CFTR activation and epithelial permeability increases. Restoring CFTR function in these diseases can restore the ability of β(2)-agonists to enhance epithelial permeability. Human bronchial epithelial cells, fully redifferentiated at the air–liquid interface, were used for (14)C mannitol flux measurements, Ussing chamber experiments, and quantitative RT-PCR. β(2)-agonists enhance epithelial permeability by activating CFTR via the β(2)-AR/adenylyl cyclase/cAMP/protein kinase A pathway. TGF-β1 inhibits β(2)-agonist–mediated CFTR activation and epithelial permeability enhancement. Although TGF-β1 down-regulates both β(2)-AR and CFTR mRNA, functionally it only decreases CFTR activity. Cigarette smoke exposure inhibits β(2)-agonist–mediated epithelial permeability increases, an effect reversed by blocking TGF-β signaling. β(2)-agonists enhance epithelial permeability via CFTR activation. TGF-β1 signaling inhibits β(2)-agonist–mediated CFTR activation and subsequent increased epithelial permeability, potentially limiting the ability of β(2)-agonists to facilitate paracellular transport in disease states unless TGF-β1 signaling is inhibited

Albuterol Modulates Its Own Transepithelial Flux via Changes in Paracellular Permeability

Although inhaled bronchodilators are commonly used in the treatment of airway disease to dilate airway smooth muscle, little is known regarding the mechanisms that regulate albuterol movement across the epithelium to reach its target, the airway smooth muscle. Because the rate of onset depends on the transepithelial transport of albuterol, to determine the mechanisms that regulate the transepithelial movement of albuterol is essential. Human bronchial epithelial cells, fully redifferentiated in culture at the air–liquid interface, were used to study the cellular uptake and total transepithelial flux of (3)H-albuterol from the apical to the basolateral surfaces. (3)H-mannitol and transepithelial electrical resistance were used to quantify changes in paracellular permeability. The majority of albuterol flux across the epithelium occurred via the paracellular route. The cellular uptake of albuterol was found to be saturable, whereas transepithelial flux was not. Cellular uptake could be inhibited by the amino acids lysine and histidine, with no effect on net transepithelial flux. Transepithelial flux was altered by maneuvers that collapsed or disrupted intercellular junctions. Acidification, usually seen in exacerbations of airway disease, decreased albuterol flux. In addition, albuterol increased its own paracellular permeability. The ability of albuterol to modulate paracellular permeability was blocked by the β(2)-adrenergic receptor–selective antagonist ICI 118551. Albuterol mainly crosses the epithelium via the paracellular pathway, but has the ability to modulate its own permeability through changes in the leakiness of tight junctions, which is modulated through the signaling of the β(2)-adrenergic receptor