Xiaohua Yang

Ineffectiveness of Tigecycline against Persistent Borrelia burgdorferi

ABSTRACT The effectiveness of a new first-in-class antibiotic, tigecycline (glycylcycline), was evaluated during the early dissemination (1 week), early immune (3 weeks), or late persistent (4 months) phases of Borrelia burgdorferi infection in C3H mice. Mice were treated with high or low doses of tigecycline, saline (negative-effect controls), or a previously published regimen of ceftriaxone (positive-effect controls). Infection status was assessed at 3 months after treatment by culture, quantitative ospA real-time PCR, and subcutaneous transplantation of joint and heart tissue into SCID mice. Tissues from all saline-treated mice were culture and ospA PCR positive, tissues from all antibiotic-treated mice were culture negative, and some of the tissues from most of the mice treated with antibiotics were ospA PCR positive, although the DNA marker load was markedly decreased compared to that in saline-treated mice. Antibiotic treatment during the early stage of infection appeared to be more effective than treatment that began during later stages of infection. The viability of noncultivable spirochetes in antibiotic-treated mice (demonstrable by PCR) was confirmed by transplantation of tissue allografts from treated mice into SCID mice, with dissemination of spirochetal DNA to multiple recipient tissues, and by xenodiagnosis, including acquisition by ticks, transmission by ticks to SCID mice, and survival through molting into nymphs and then into adults. Furthermore, PCR-positive heart base tissue from antibiotic-treated mice revealed RNA transcription of several B. burgdorferi genes. These results extended previous studies with ceftriaxone, indicating that antibiotic treatment is unable to clear persisting spirochetes, which remain viable and infectious, but are nondividing or slowly dividing.

Antialarmin Effect of Tick Saliva during the Transmission of Lyme Disease

ABSTRACT Tick saliva has potent immunomodulatory properties. In arthropod-borne diseases, this effect is largely used by microorganisms to increase their pathogenicity and to evade host immune responses. We show that in Lyme borreliosis, tick salivary gland extract and a tick saliva protein, Salp15, inhibit in vitro keratinocyte inflammation induced by Borrelia burgdorferi sensu stricto or by the major outer surface lipoprotein of Borrelia, OspC. Chemokines (interleukin-8 [IL-8] and monocyte chemoattractant protein 1 [MCP-1]) and several antimicrobial peptides (defensins, cathelicidin, psoriasin, and RNase 7) were downregulated. Interestingly, antimicrobial peptides (AMPs) transiently inhibited bacterial motility but did not kill the organisms when tested in vitro. We conclude that tick saliva affects the chemotactic properties of chemokines and AMPs on immune cells and has an antialarmin effect on human primary keratinocytes. Alarmins are mediators that mobilize and activate antigen-presenting cells. Inhibition of cutaneous innate immunity and of the migration of immune cells to the site of the tick bite ensures a favorable environment for Borrelia. The bacterium can then multiply locally and, subsequently, disseminate to the target organs, including joints, heart, and the central nervous system.

A New Approach to a Lyme Disease Vaccine

A single recombinant outer surface protein A (OspA) antigen designed to contain protective elements from 2 different OspA serotypes (1 and 2) is able to induce antibody responses that protect mice against infection with either Borrelia burgdorferi sensu stricto (OspA serotype-1) or Borrelia afzelii (OspA serotype-2). Protection against infection with B burgdorferi ss strain ZS7 was demonstrated in a needle-challenge model. Protection against B. afzelii species was shown in a tick-challenge model using feral ticks. In both models, as little as .03 μg of antigen, when administered in a 2-dose immunization schedule with aluminum hydroxide as adjuvant, was sufficient to provide complete protection against the species targeted. This proof of principle study proves that knowledge of protective epitopes can be used for the rational design of effective, genetically modified vaccines requiring fewer OspA antigens and suggests that this approach may facilitate the development of an OspA vaccine for global use.

Defensin Is Suppressed by Tick Salivary Gland Extract During the In Vitro Interaction of Resident Skin Cells with Borrelia burgdorferi

TO THE EDITOR The causative agent of Lyme disease, Borrelia burgdorferi, is a spirochete parasitizing vertebrates and transmitted by the tick, Ixodes sp. Lyme disease is the most common vector-borne disease in the Northern Hemisphere (Piesman and Gern, 2004). It manifests itself frequently with an erythema migrans rash at the site of the infection. However, the infection can progress and disseminate, affecting other skin sites, the joints, the heart, and the nervous system in humans. Control of the initial cutaneous phase may play a crucial role in the outcome of the disease, as not all patients develop disseminated clinical infection (Steere and Glickstein, 2004). The skin constitutes a complex physical barrier. First, the epidermis comprises mainly keratinocytes (KCs) and Langerhans cells. These cells possess specific Toll-like receptors (TLRs) that recognize certain defined patterns, the pathogen-associated molecular patterns, present on pathogens. The interaction between pathogen-associated molecular patterns and TLRs leads to the activation of the NF-kB pathway, with the production of inflammatory molecules including chemokines, cytokines, and antimicrobial peptides (AMPs) (Pivarcsi et al., 2004). These molecules of innate immunity are essential to the control of infection. Dermal fibroblasts (FBs) constitute a second group of resident skin cells that secrete the extracellular matrix and communicate with other cell types such as dermal dendritic cells, mast cells, macrophages, and KCs (Sorrell and Caplan, 2004). Ebnet et al. (1997) reported the activation of NF-kB upon the interaction of Borrelia with human KCs and FBs. However, no recent study has investigated the potential induction of AMPs in the interaction of B. burgdorferi with resident skin cells. We studied the induction of the proinflammatory molecule IL-8, defensins, and cathelicidin. IL-8 is a chemokine that attracts neutrophils, major cells involved in inflammation and secreting AMPs. Defensins are produced by leukocytes and various epithelial cells (Ganz, 2003). The cathelicidin LL-37, in humans, is present constitutively in neutrophil granules and is inducible in epithelial cells in response to infection. Their role in the control of several skin inflammations is well established (Nizet et al., 2001; Ong et al., 2002; Braff et al., 2005). We used ELISA to measure the secretion of IL-8 and HBD-2 (human bdefensin-2) and quantitative real-time reverse transcriptase-PCR to study the induction of HBD-2 and LL-37 mRNAs by KCs and FBs. IL-8 was induced by B. burgdorferi in a dose-dependent manner in KCs and FBs (Figure 1a and b). Regarding AMP mRNAs, defensin, but not cathelicidin, was induced in KCs (Figure 1c and f). In FBs, Borrelia did not induce HBD-2 and a weak expression of LL-37 mRNA was observed (Figure 1d and f). A kinetic study revealed that IL-8 secretion reached its highest level at 12 hours for both KCs and FBs (Figure 1g and h), whereas HBD-2 mRNA reached a peak at 6 hours in KCs (Figure 1i), also confirmed by the measure of HBD-2 secretion by ELISA (data not shown). Only very weak amounts were found to be secreted by FBs upon stimulation with Borrelia (Figure 1j). For LL-37 mRNA, only marginal expression by KCs was shown (Figure 1k), whereas Borrelia elicited a much higher expression in FBs, which peaked at 6 hours (Figure 1l). As Borrelia are transmitted by the Ixodes tick, we analyzed the effect of tick salivary gland (SG) extract on host cell inflammation. As can be seen in Figure 2, induction of IL-8 and HBD-2 was significantly inhibited by SG extract. The inhibitory effect on IL-8 Abbreviations: AMP, antimicrobial peptide; FB, fibroblast; HBD, human b-defensin; KC, keratinocyte; SG, salivary gland; TLR, Toll-like receptor

Proteome analysis of Borrelia burgdorferi response to environmental change.

We examined global changes in protein expression in the B31 strain of Borrelia burgdorferi, in response to two environmental cues (pH and temperature) chosen for their reported similarity to those encountered at different stages of the organisms life cycle. Multidimensional nano-liquid chromatographic separations coupled with tandem mass spectrometry were used to examine the array of proteins (i.e., the proteome) of B. burgdorferi for different pH and temperature culture conditions. Changes in pH and temperature elicited in vitro adaptations of this spirochete known to cause Lyme disease and led to alterations in protein expression that are associated with increased microbial pathogenesis. We identified 1,031 proteins that represent 59% of the annotated genome of B. burgdorferi and elucidated a core proteome of 414 proteins that were present in all environmental conditions investigated. Observed changes in protein abundances indicated varied replicon usage, as well as proteome functional distributions between the in vitro cell culture conditions. Surprisingly, the pH and temperature conditions that mimicked B. burgdorferi residing in the gut of a fed tick showed a marked reduction in protein diversity. Additionally, the results provide us with leading candidates for exploring how B. burgdorferi adapts to and is able to survive in a wide variety of environmental conditions and lay a foundation for planned in situ studies of B. burgdorferi isolated from the tick midgut and infected animals.

In vitro activity of tigecycline against multiple strains of Borrelia burgdorferi.

OBJECTIVES To compare the antimicrobial activity of tigecycline and doxycycline against multiple isolates of Borrelia burgdorferi. METHODS In vitro antimicrobial assays were carried out using a microdilution assay. The time needed to inhibit, immobilize and kill the B31 strain of B. burgdorferi was determined. The MIC, MBC and concentration needed to immobilize the organism were determined for each antimicrobial for various strains of B. burgdorferi. RESULTS Tigecycline inhibited the growth of and killed the organism more rapidly than doxycycline. Tigecycline was able to kill B. burgdorferi within 24 h at clinically achievable concentrations (<1 mg/L). In contrast, doxycycline was bacteriostatic and required 48-72 h to achieve its maximal inhibitory effect. The anti-Borrelia activity of the antibiotics was tested against 20 different isolates from three species. Tigecycline was 16- to 1000-fold more active than doxycycline at immobilizing Borrelia for the 20 isolates tested. CONCLUSIONS We demonstrate that the in vitro activity of tigecycline against B. burgdorferi is superior to that of doxycycline. Tigecycline acted more rapidly and was bactericidal, whereas doxycycline was bacteriostatic and required a more prolonged co-incubation to achieve its maximal inhibitory effect.

An epigenome-wide DNA methylation study of PTSD and depression in World Trade Center responders

Previous epigenome-wide association studies (EWAS) of posttraumatic stress disorder (PTSD) and major depressive disorder (MDD) have been inconsistent. This may be due to small sample sizes, and measurement and tissue differences. The current two EWA analyses of 473 World Trade Center responders are the largest to date for both PTSD and MDD. These analyses investigated DNA methylation patterns and biological pathways influenced by differentially methylated genes associated with each disorder. Methylation was profiled on blood samples using Illumina 450 K Beadchip. Two EWA analyses compared current versus never PTSD, and current versus never MDD, adjusting for cell types and demographic confounders. Pathway and gene set enrichment analyses were performed to understand the complex biological systems of PTSD and MDD. No significant epigenome-wide associations were found for PTSD or MDD at an FDR P<0.05. The majority of genes with differential methylation at a suggestive threshold did not overlap between the two disorders. Pathways significant in PTSD included a regulator of synaptic plasticity, oxytocin signaling, cholinergic synapse and inflammatory disease pathways, while only phosphatidylinositol signaling and cell cycle pathways emerged in MDD. The failure of the current EWA analyses to detect significant epigenome-wide associations is in contrast with disparate findings from previous, smaller EWA and candidate gene studies of PTSD and MDD. Enriched gene sets involved in several biological pathways, including stress response, inflammation and physical health, were identified in PTSD, supporting the view that multiple genes play a role in this complex disorder.