Anti-tuberculosis activity of α-helical antimicrobial peptides: de novo designed L- and D-enantiomers versus L- and D-LL-37.

With the emergence of multi-drug resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis (Mtb), a new class of antimycobacterial agents with very different modes of action compared to classical antibiotics, are urgently needed. In this study, a series of 26-residue, amphipathic, α-helical antimicrobial peptides consisting of all D-amino acid residues and synthetic human L-LL37 (L-enantiomer) and D-LL37 (D-enantiomer) were investigated against M. tuberculosis susceptible strain (H37Rv) and a clinical multi-drug resistant strain (Vertulo). Minimal inhibitory concentrations (MICs) were determined through a peptide killing assay. D5, the most active analog against M. tuberculosis had a MIC value of 11.2 µM (35.2 µg/ml) against H37Rv strain and 15.6 µM (49 µg/ml) against the MDR strain. Peptide D1 had similar activity as D5 against the MDR strain (57 µg/mL), a 9-fold improvement in hemolytic activity and a 7.4-fold better therapeutic index compared to D5. Surprisingly, LL37 enantiomers showed little to no activity compared to the de-novo designed α-helical antimicrobial peptides.

Dry powder measles vaccine: particle deposition, virus replication, and immune response in cotton rats following inhalation.

A stable and high potency dry powder measles vaccine with a particle size distribution suitable for inhalation was manufactured by CO(2)-Assisted Nebulization with a Bubble Dryer(®) (CAN-BD) process from bulk liquid Edmonston-Zagreb live attenuated measles virus vaccine supplied by the Serum Institute of India. A novel dry powder inhaler, the PuffHaler(®) was adapted for use in evaluating the utility of cotton rats to study the vaccine deposition, vaccine virus replication, and immune response following inhalation of the dry powder measles vaccine. Vaccine deposition in the lungs of cotton rats and subsequent viral replication was detected by measles-specific RT-PCR, and viral replication was confined to the lungs. Inhalation delivery resulted in an immune response comparable to that following injection. The cotton rat model is useful for evaluating new measles vaccine formulations and delivery devices.

The roles of antimicrobial peptides in innate host defense.

Antimicrobial peptides (AMPs) are multi-functional peptides whose fundamental biological role in vivo has been proposed to be the elimination of pathogenic microorganisms, including Gram-positive and -negative bacteria, fungi, and viruses. Genes encoding these peptides are expressed in a variety of cells in the host, including circulating phagocytic cells and mucosal epithelial cells, demonstrating a wide range of utility in the innate immune system. Expression of these genes is tightly regulated; they are induced by pathogens and cytokines as part of the host defense response, and they can be suppressed by bacterial virulence factors and environmental factors which can lead to increased susceptibility to infection. New research has also cast light on alternative functionalities, including immunomodulatory activities, which are related to their unique structural characteristics. These peptides represent not only an important component of innate host defense against microbial colonization and a link between innate and adaptive immunity, but also form a foundation for the development of new therapeutic agents.

Nanomedicine for respiratory diseases.

Treatment of respiratory diseases and infections has proved to be a challenging task, with the incidence of these ailments increasing worldwide. Nanotechnology-based drug and gene delivery systems offer a possible solution to some of the shortfalls of the current treatment regimen. Nanobased drug delivery systems have revolutionised the field of pharmacotherapy by presenting the ability to alter the pharmacokinetics of the conventional drugs to extend the drug retention time, reduce the toxicity and increase the half-life of the drugs. Delivery of exogenous genes to the airway epithelium in vivo has been limited by several physiological barriers, resulting in the low success rate of these systems. With the advent of nanotechnology, DNA compacted with cationic polymers to produce nanoparticles has exhibited a significant increase in the transfection efficiencies. With nanoparticulate drug/gene delivery systems, specific cells can be targeted by functionalising the polymeric nanoparticles with ligands that allow the particles to dock at a specific site of the cell. In addition, polymeric systems allow for the cargo to be released in a controlled and stimuli-responsive manner. The advantages that nanoparticulate delivery systems present in the treatment of respiratory diseases and infections are summarised in this review.

Hairpin ribozyme-antisense RNA constructs can act as molecular Lassos.

We have developed a novel class of antisense agents, RNA Lassos, which are capable of binding to and circularizing around complementary target RNAs. The RNA Lasso consists of a fixed sequence derived from the hairpin ribozyme and an antisense segment whose size and sequence can be varied to base pair with accessible sites in the target RNA. The ribozyme catalyzes self-processing of the 5'- and 3'-ends of a transcribed Lasso precursor and ligates the processed ends to produce a circular RNA. The circular and linear forms of the self-processed Lasso coexist in an equilibrium that is dependent on both the Lasso sequence and the solution conditions. Lassos form strong, noncovalent complexes with linear target RNAs and form true topological linkages with circular targets. Lasso complexes with linear RNA targets were detected by denaturing gel electrophoresis and were found to be more stable than ordinary RNA duplexes. We show that expression of a fusion mRNA consisting of a sequence from the murine tumor necrosis factor-alpha (TNF-alpha) gene linked to luciferase reporter can be specifically and efficiently blocked by an anti-TNF Lasso. We also show in cell culture experiments that Lassos directed against Fas pre-mRNA were able to induce a change in alternative splicing patterns.

Defective killing of Staphylococcus aureus in atopic dermatitis is associated with reduced mobilization of human beta-defensin-3.

BACKGROUND: Individuals with atopic dermatitis (AD) have frequent colonization and infection with Staphylococcus aureus. Rapid elimination of S. aureus depends on constitutive synthesis and mobilization of human beta-defensin-3 (HBD-3). OBJECTIVE: To determine whether keratinocytes in AD, compared with normal, skin are less able to kill S. aureus rapidly, and to assess the potential role that abnormally low mobilization of HBD-3 onto S. aureus has in this process. METHODS: Skin samples from 10 normal individuals and 10 patients with AD were compared for synthesis and mobilization of HBD-3 onto surface-associated S. aureus. Furthermore, keratinocytes from 10 individuals were studied for the effects of T(H)2 cytokines on the ability of the cells to synthesize and mobilize HBD-3, and to kill S. aureus. RESULTS: Keratinocytes in skin biopsies from subjects with AD were defective in killing S. aureus relative to normal individuals (P < .001). The constitutive levels of HBD-3 in the epidermal keratinocytes were similar between normal individuals and those with AD. However, the cells of patients with AD were unable to mobilize HBD-3 efficiently to kill S. aureus. Physiologic Ca(++) was essential for development of normal HBD-3 levels by cultured human keratinocytes. Mobilization of HBD-3 and the ability to kill S. aureus were significantly (P < .05) inhibited by IL-4 and IL-13. Antagonism of IL-4/10/13 with antibodies significantly (P < .01) improved mobilization of HBD-3 onto the surface of S. aureus by skin from patients with AD. CONCLUSION: Patients with AD have problems with S. aureus skin infection. This is a result of increased levels of T(H)2 cytokines, which inhibit keratinocyte mobilization of HBD-3.

The constitutive capacity of human keratinocytes to kill Staphylococcus aureus is dependent on beta-defensin 3.

Normal skin is often exposed to bacteria, including potent pathogens such as E. coli, Staphylococcus aureus, and Streptococcus sp., but these microbes usually do not cause skin inflammation or infection in healthy individuals. Therefore, we hypothesized that there must be a constitutive mechanism for rapid destruction and elimination of small numbers of bacteria which penetrate the stratum corneum from everyday activities. This study found that exposure of keratinocytes cultured from a number of individuals to S. aureus resulted in approximately 2-3 log better killing than by HaCaT cells within 1 hour. Killing required contact between the keratinocytes and the bacteria, but was not dependent on internalization. Contact between the bacteria and the keratinocytes resulted in rapid deposition of several antimicrobial peptides onto the bacteria, but only human beta-defensin (HBD) 3 accumulated at levels sufficient to account for killing when S. aureus were exposed to human skin explants. Blocking peptide binding of HBD3 inhibited killing of the bacteria, indicating an essential role for beta-defensin 3 in the constitutive killing of bacteria by normal keratinocytes.

Macrophage inflammatory protein 3alpha deficiency in atopic dermatitis skin and role in innate immune response to vaccinia virus.

BACKGROUND: Patients with atopic dermatitis (AD) are prone to disseminated viral skin infections and therefore are not vaccinated against smallpox because of potential complications. Macrophage inflammatory protein 3alpha (MIP-3alpha) is a C-C chemokine expressed by keratinocytes that exhibits antimicrobial activity against bacteria and fungi; however, its role in antiviral innate immunity is unknown. OBJECTIVE: Evaluate the level of MIP-3alpha in AD skin and its role in the innate immune response to vaccinia virus (VV). METHODS: Macrophage inflammatory protein 3alpha levels were evaluated using real-time RT-PCR, immunodot-blot, and immunohistochemistry. The antiviral activity of MIP-3alpha was determined using a standard viral plaque assay. RESULTS: Macrophage inflammatory protein 3alpha gene expression was significantly (P < .01) decreased in AD skin (0.21 +/- 0.05 ng MIP-3alpha/ng glyceraldehyde-3-phosphate dehydrogenase) compared with psoriasis skin (0.67 +/- 0.13). This was confirmed at the protein level using immunohistochemistry. We further demonstrate that T(H)2 cytokines downregulate MIP-3alpha expression. The importance of MIP-3alpha in the innate immune response against VV was established by first demonstrating that MIP-3alpha exhibits activity against VV. Second, VV replication was significantly increased (P < .01) in keratinocytes treated with an antibody to neutralize MIP-3alpha. CONCLUSION: The current study demonstrates that MIP-3alpha exhibits antiviral activity against VV and demonstrates the importance of MIP-3alpha in the innate immune response against VV. In addition, AD skin is deficient in MIP-3alpha, in part because of the overexpression of T(H)2 cytokines in AD skin. CLINICAL IMPLICATIONS: MIP-3alpha deficiency in AD skin contributes to patients' increased propensity toward eczema vaccinatum. Increasing MIP-3alpha or neutralizing T(H)2 cytokines could prevent adverse reactions in patients with AD after smallpox vaccination.

The potential advantages of nanoparticle drug delivery systems in chemotherapy of tuberculosis.

Nanoparticle-based drug delivery systems have considerable potential for treatment of tuberculosis (TB). The important technological advantages of nanoparticles used as drug carriers are high stability, high carrier capacity, feasibility of incorporation of both hydrophilic and hydrophobic substances, and feasibility of variable routes of administration, including oral application and inhalation. Nanoparticles can also be designed to allow controlled (sustained) drug release from the matrix. These properties of nanoparticles enable improvement of drug bioavailability and reduction of the dosing frequency, and may resolve the problem of nonadherence to prescribed therapy, which is one of the major obstacles in the control of TB epidemics. This article highlights some of the issues of nanotechnology relevant to the anti-TB drugs.

Selective killing of vaccinia virus by LL-37: implications for eczema vaccinatum.

Possible bioterrorism with smallpox has led to the resumption of smallpox (vaccinia virus) immunization. One complication, eczema vaccinatum, occurs primarily in patients with atopic dermatitis (AD). Skin lesions of patients with AD, but not psoriasis, is deficient in the cathelicidin antimicrobial peptide (LL-37) and human beta-defensin-2 (HBD-2). We hypothesized that this defect may explain the susceptibility of patients with AD to eczema vaccinatum. The Wyeth vaccine strain of vaccinia virus was incubated with varying concentrations of human (LL-37) and murine (CRAMP) cathelicidins, human alpha-defensin (HBD-1, HBD-2), and a control peptide. Outcomes included quantification of viral PFU, vaccinia viral gene expression by quantitative real-time RT-PCR, and changes in virion structure by transmission electron microscopy. CRAMP knockout mice and control animals were inoculated by skin pricks with 2 x 10(5) PFU of vaccinia and examined daily for pox development. Physiologic amounts of human and murine cathelicidins (10-50 micro M), but not human defensins, which had antibacterial activity, resulted in the in vitro reduction of vaccinia viral plaque formation (p < 0.0001), vaccinia mRNA expression (p < 0.001), and alteration of vaccinia virion structure. In vivo vaccinia pox formation occurred in four of six CRAMP knockout animals and in only one of 15 control mice (p < 0.01). These data support a role for cathelicidins in the inhibition of orthopox virus (vaccinia) replication both in vitro and in vivo. Susceptibility of patients with AD to eczema vaccinatum may be due to a deficiency of cathelicidin.

Serine-arginine-rich protein p30 directs alternative splicing of glucocorticoid receptor pre-mRNA to glucocorticoid receptor beta in neutrophils.

Glucocorticoid (GC) insensitivity is a major clinical challenge in the treatment of many inflammatory diseases. It has been shown previously that GC insensitivity, in several inflammatory cell types, is due to an overabundance of the beta isoform of the glucocorticoid receptor (GCRbeta) relative to the ligand binding isoform, GCRalpha. GCRbeta functions as a dominant inhibitor of GCRalpha action. A number of GCR isoforms are created from the same pre-mRNA transcript via alternative splicing, and the factor or factors that control alternative splicing of GCR pre-mRNA are of great importance. In the current study, we have identified the predominant alternative splicing factor present in human neutrophils, which is known to be exceptionally GC-insensitive. The predominant alternative splicing factor in neutrophils is SRp30c, which is one of several highly conserved serine-arginine-rich (SR) proteins that are involved in both constitutive and alternative splicing in eukaryotic cells. Inhibition of SRp30c expression with antisense oligonucleotide strongly inhibited expression of GCRbeta and stimulated expression of GCRalpha. Antisense molecules targeted to other SR proteins had no effect. Our data indicate that SRp30c is necessary for alternative splicing of the GCR pre-mRNA to create mRNA encoding GCRbeta.

A role for STAT5 in the pathogenesis of IL-2-induced glucocorticoid resistance.

Glucocorticoids (GC) are highly effective in the control of diseases associated with T cell activation. However, a subset of individuals is GC insensitive. Previous studies have demonstrated that IL-2 can induce steroid resistance in mouse T cells. However, the mechanism for this phenomenon is unknown. In the current study we found that the murine cell line (HT-2) is steroid resistant when incubated with IL-2, but steroid sensitive when grown in IL-4. Furthermore, when HT-2 cells are treated with IL-2, the glucocorticoid receptor (GCR) does not translocate to the cell nucleus after dexamethasone treatment. In contrast, the GCR in IL-4-stimulated HT-2 cells does translocate into the cell nucleus after dexamethasone treatment. IL-2-induced steroid insensitivity in HT-2 cells appears to be a signaling event as the effects of IL-2 on nuclear translocation of the GCR occurred within 30 min even in the presence of cycloheximide. Indeed, preincubation of HT-2 cells with a Janus-associated kinase 3 inhibitor restored nuclear translocation of the GCR even in the presence of IL-2. Immunoprecipitation experiments revealed that phosphorylated STAT5 and GCR formed immune complexes. This association may lead to retardation of GCR nuclear translocation because IL-2 was not able to induce steroid insensitivity in splenocytes from STAT5 knockout mice. This study demonstrates a novel role for STAT5 in IL-2-induced steroid insensitivity.

Increased glucocorticoid receptor Beta expression converts mouse hybridoma cells to a corticosteroid-insensitive phenotype.

Glucocorticoid (GC) insensitivity is a challenging clinical problem associated with many chronic inflammatory disorders and life-threatening disease progression. The molecular basis of GC insensitivity, however, is unknown. Alternative splicing of the GC receptor (GCR) pre-mRNA generates a second GCR, termed GCRbeta, which does not bind GC but antagonizes the transactivating activity of the classic GCR, termed GCRalpha. GC-insensitive conditions have been associated with increased GCRbeta expression. Whether or not increased GCRbeta expression can contribute to GC insensitivity, however, remains controversial. To more precisely demonstrate the effect of GCRbeta on steroid responsiveness, we virally transduced GCRbeta cDNA into mouse DO-11.10 hybridoma cells, as mice are known to be deficient in the GCRbeta gene. We demonstrate that viral transduction of GCRbeta cDNA into mouse hybridoma cells to induce stable expression of GCRbeta results in GC insensitivity of these cells. Furthermore, in such cells GCRalpha is complexed with GCRbeta. Such heterodimer formation may account for the reduced effectiveness of GC action in cells overexpressing GCRbeta.

Tumor necrosis factor alpha stimulates killing of Mycobacterium tuberculosis by human neutrophils.

The ability of human neutrophils to aid in defense against pulmonary infection with Mycobacterium tuberculosis is controversial. In this study, we have shown that neutrophils respond to and phagocytose M. tuberculosis in human lesions. Neutrophils from healthy individuals were able to kill significant fractions of an inoculum of M. tuberculosis within 1 h of phagocytosis, and this ability was enhanced by tumor necrosis factor alpha but not by gamma interferon. The mycobactericidal mechanism was nonoxidative, as inhibitors of reactive oxygen or reactive nitrogen intermediates did not interfere with killing. However, the mycobactericidal mechanism was associated with increased exposure of intracellular M. tuberculosis to neutrophil defensins. In vitro, human neutrophil peptides 1 to 3 were not able to kill the bacilli even at much higher levels. These studies support the concept that human neutrophils are directly involved in defense against infection with M. tuberculosis.