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.

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.

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 β-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 α-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 × 105 PFU of vaccinia and examined daily for pox development. Physiologic amounts of human and murine cathelicidins (10–50 μM), 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.

Neutrophils are markedly less sensitive to glucocorticoids than T cells, making it difficult to control inflammation in neutrophil-mediated diseases. Development of new antiinflammatory strategies for such diseases would be aided by an understanding of mechanisms underlying differential steroid responsiveness. Two protein isoforms of the human glucocorticoid receptor (GR) exist, GRα and GRβ, which arise from alternative splicing of the GR pre-mRNA primary transcripts. GRβ does not bind glucocorticoids and is an inhibitor of GRα activity. Relative amounts of these two GRs can therefore determine the level of glucocorticoid sensitivity. In this study, human neutrophils and peripheral blood mononuclear cells (PBMCs) were studied to determine the relative amounts of each GR isoform.The mean fluorescence intensity (MFI) using immunofluorescence analysis for GRα was 475 ± 62 and 985 ± 107 for PBMCs and neutrophils, respectively. For GRβ, the MFI was 350 ± 60 and 1,389 ± 143 for PBMCs and neutrophils, respectively (P < 0.05). After interleukin (IL)-8 stimulation of neutrophils, there was a statistically significant increase in intensity of GRβ staining to 2,497 ± 140 (P < 0.05). No change in GRα expression was observed. This inversion of the GRα/GRβ ratio in human neutrophils compared with PBMCs was confirmed by quantitative Western analysis. Increased GRβ mRNA expression in neutrophils at baseline, and after IL-8 exposure, was observed using RNA dot blot analysis. Increased levels of GRα/GRβ heterodimers were found in neutrophils as compared with PBMCs using coimmunoprecipitation/Western analysis. Transfection of mouse neutrophils, which do not contain GRβ, resulted in a significant reduction in the rate of cell death when treated with dexamethasone.We conclude that high constitutive expression of GRβ by human neutrophils may provide a mechanism by which these cells escape glucocorticoid-induced cell death. Moreover, upregulation of this GR by proinflammatory cytokines such as IL-8 further enhances their survival in the presence of glucocorticoids during inflammation.

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.

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.The observation that humans are not uniformly susceptible to infection by Mycobacterium tuberculosis may imply the existence of resistance mechanisms capable of sterilizing inhaled inoculae which operate independently of acquired immunity. Although studies with rodent models indicate that alveolar macrophages are important for containment of experimental aerogenic infections, it may be necessary to look to other effector cells to understand innate immune mechanisms which protect against human infections.There have been two reports that human neutrophils can kill virulent M. tuberculosis in vitro (5, 18). However, more-recent examinations of this issue have been unable to confirm these results (2, 10). Majeed et al. were able to demonstrate that cultured human neutrophils were able to kill an attenuated strain of M. tuberculosis, H37Ra, in a calcium-dependent manner (21). However, as H37Ra is not known to be virulent for humans, it remains important to resolve the issue of neutrophil capacity to kill virulent M. tuberculosis. Pedrosa et al. have suggested that murine neutrophils are protective against experimental infections of mice, although they found no evidence that the neutrophils phagocytosed the tubercle bacilli (27).The goal of these studies was to understand whether the neutrophil response characteristic of the earliest responses to tuberculosis (TB) infection could be effective in reducing or eliminating the inoculum. To this end, we established a model of in vitro infection of human neutrophils and characterized some of the effector mechanisms which come into play during the response of neutrophils both in vitro and during pulmonary infection of humans. We show that neutrophils in human pulmonary lesions contain intracellular M. tuberculosis, confirming a phagocytic role for neutrophils in human TB. We also show that human neutrophils can kill virulent intracellular M. tuberculosis in vitro, but with variability between individuals. Mycobactericidal activity could be stimulated by exposure of infected neutrophils to tumor necrosis facto...

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