James P. Chambers

A review of molecular recognition technologies for detection of biological threat agents

The present review summarizes the state of the art in molecular recognition of biowarfare agents and other pathogens and emphasizes the advantages of using particular types of reagents for a given target (e.g. detection of bacteria using antibodies versus nucleic acid probes). It is difficult to draw firm conclusions as to type of biorecognition molecule to use for a given analyte. However, the detection method and reagents are generally target-driven and the user must decide on what level (genetic versus phenotypic) the detection should be performed. In general, nucleic acid-based detection is more specific and sensitive than immunological-based detection, while the latter is faster and more robust. This review also points out the challenges faced by military and civilian defense components in the rapid and accurate detection and identification of harmful agents in the field. Although new and improved sensors will continue to be developed, the more crucial need in any biosensor may be the molecular recognition component (e.g. antibody, aptamer, enzyme, nucleic acid, receptor, etc.). Improvements in the affinity, specificity and mass production of the molecular recognition components may ultimately dictate the success or failure of detection technologies in both a technical and commercial sense. Achieving the ultimate goal of giving the individual soldier on the battlefield or civilian responders to an urban biological attack or epidemic, a miniature, sensitive and accurate biosensor may depend as much on molecular biology and molecular engineering as on hardware engineering. Fortunately, as this review illustrates, a great deal of scientific attention has and is currently being given to the area of molecular recognition components. Highly sensitive and specific detection of pathogenic bacteria and viruses has increased with the proliferation of nucleic acid and immuno-based detection technologies. If recent scientific progress is a fair indicator, the future promises remarkable new developments in molecular recognition elements for use in biosensors with a vast array of applications.

Production of Tumor Necrosis Factor-alpha, Interleukin 1-beta, Interleukin 2, and Interleukin 6 by rat leukocyte subpopulations after exposure to Substance P

The interaction between components of the nervous system and multiple target cells in the cutaneous immune system has been receiving increasing attention. Recently, the involvement of neuropeptides has been demonstrated to play an important role in the inflammatory cascade. Neuropeptides such as Substance P are released by cutaneous neurons and modulate the function of immunocompetent and inflammatory cells as well as epithelial and endothelial cells. Substance P has been shown to function as a mediator for cell proliferation, cytokine production, and as an upregulator of various cell surface receptors. In this study, we show the effect of Substance P on the production of Tumor Necrosis Factor-alpha, Interleukin 1-beta, Interleukin 2, and Interleukin 6 by T-lymphocytes, macrophages and neutrophils. These data demonstrate that pathophysiological levels of Substance P induce production of cytokines in all three cell populations tested. Interestingly, T-cells demonstrated the highest percentage of cells expressing all four cytokines. In contrast, macrophages and neutrophils produced the highest absolute levels of cytokines. The elucidation of mediating mechanisms of Substance P activation of leukocytes is crucial to the understanding of the cutaneous inflammatory cascade and involvement of the peripheral nervous system on the immune system. These findings suggest that Substance P participates in the complex network of mediators that regulate cutaneous inflammation and potentially the rate of wound healing.

Intranasal vaccination with a secreted chlamydial protein enhances resolution of genital Chlamydia muridarum infection, protects against oviduct pathology, and is highly dependent upon endogenous gamma interferon production.

ABSTRACT There is currently no licensed vaccine against Chlamydia trachomatis, the leading cause of sexually transmitted bacterial disease worldwide. Conventional vaccination attempts using surface-exposed chlamydial antigens have achieved only partial success. We have employed a novel vaccination strategy using a secreted protein, chlamydial protease-like activity factor (CPAF), which has been shown to degrade host major histocompatibility complex transcription factors and keratin-8 and therefore may allow immune evasion and establishment of a productive infection. Intranasal immunization using recombinant CPAF (rCPAF) plus interleukin-12 (IL-12) (rCPAF+IL-12 immunization) was used to assess the protective immunity against genital Chlamydia muridarum infection in BALB/c mice. rCPAF+IL-12 immunization induced robust gamma interferon (IFN-γ) production and minimal IL-4 production by splenocytes upon in vitro recall with rCPAF. The total and immunoglobulin G2a (IgG2a) anti-rCPAF antibody levels in serum were significantly elevated after rCPAF+IL-12 vaccination, as were the total antibody, IgG2a, and IgA levels in bronchoalveolar lavage and vaginal fluids when the animals were compared to animals that received rCPAF alone. rCPAF+IL-12-vaccinated mice displayed significantly reduced bacterial shedding upon chlamydial challenge and accelerated resolution of infection compared to mock-immunized (phosphate-buffered saline) animals. Moreover, rCPAF+IL-12-immunized animals exhibited protection against pathological consequences of chlamydial infection, including the development of hydrosalpinx and oviduct dilatation. This vaccination regimen also reduced the development of fibrosis and the influx of neutrophils into the upper genital tract when the animals were compared to mock-immunized (phosphate-buffered saline) animals after bacterial challenge. rCPAF+IL-12-mediated resolution of the bacterial infection and protection against Chlamydia-induced inflammatory disease were highly dependent on endogenous IFN-γ production. Together, these results demonstrate that secreted chlamydial antigens may be novel vaccine candidates to induce protective immunity.

Exogenous administration of substance P enhances wound healing in a novel skin-injury model

Soft tissue injury accounts for approximately 44% of all wounds in both the military and civilian populations. Following injury to soft tissue, Substance P (SP) and other neuropeptides are released by cutaneous neurons and modulate the function of immunocompetent and inflammatory cells, as well as epithelial and endothelial cells. The interaction between these components of the nervous system and multiple target cells affecting cutaneous repair is of increasing interest. In this report, we describe the effects of SP on wound repair in a novel, laser-induced, skin-wound model. Gross and histologic examination of laser-induced injury revealed that exogenously administered SP affects wound healing via neurite outgrowth, in addition to adhesion molecule and neurokinin-1 receptor involvement in vivo. All SP effects were decreased by pretreatment with Spantide II, an SP antagonist. The elucidation of SP-mediating mechanisms is crucial to firmly establishing the involvement and interaction of the peripheral nervous system and the immune system in cutaneous repair. Findings presented here suggest that SP participates in the complex network of mediators involved in cutaneous inflammation and wound healing.

In vivo and in vitro effects of the pineal gland and melatonin on [Ca2++ Mg2+]‐dependent ATPase in cardiac sarcolemma

Abstract: The possible diurnal variation in cardiac [Ca2++ Mg2+]‐dependent ATPase (Ca2+ pump) activity and the influence of pinealectomy and melatonin on this enzyme in rat heart have been studied. Lowest levels of cardiac sarcolemma] membrane [Ca2++ Mg2+]‐dependent ATPase activity were measured in late afternoon in rats kept under a 14:10 light:dark cycle. Late in the dark phase the enzyme activity began to increase with the rise continuing until 0900, 3 hr after light onset. These time‐dependent changes in [Ca2++ Mg2+]‐dependent ATPase activity did not occur in either pinealectomized or light‐exposed rats suggesting that melatonin, secreted from the pineal gland during the night, induces the change in [Ca2++ Mg2+]‐dependent ATPase activity. In vitro studies in which cardiac tissue was incubated in the presence of melatonin over a wide range of doses showed that this indole stimulated the Ca2+ pump. The half‐maximal effect of melatonin was observed at a melatonin concentration of 28 ng/ml. These findings suggest that the daily change in [Ca2++ Mg2+]‐dependent ATPase activity in the sarcolemma of heart tissue is a result of the circadian rhythm in pineal melatonin production and secretion. These findings may be applicable to normal cardiac physiology.

Next-Generation Ion Torrent Sequencing of Drug Resistance Mutations in Mycobacterium tuberculosis Strains

ABSTRACT A novel protocol for full-length Mycobacterium tuberculosis gene analysis of first- and second-line drug resistance was developed using the Ion Torrent Personal Genome Machine (PGM). Five genes—rpoB (rifampin), katG (isoniazid), pncA (pyrazinamide), gyrA (ofloxacin/fluoroquinolone), and rrs (aminoglycosides)—were amplified and sequenced, and results were compared to those obtained by genotypic Hain line probe assay (LPA) and phenotypic Bactec MGIT 960 analysis using 26 geographically diverse South African clinical isolates collected between July and November 2011. Ion Torrent sequencing exhibited 100% (26/26) concordance to phenotypic resistance obtained by MGIT 960 culture and genotypic rpoB and katG results by LPA. In several rifampin-resistant isolates, Ion Torrent sequencing revealed uncommon substitutions (H526R and D516G) that did not have a defined mutation by LPA. Importantly, previously uncharacterized mutations in rpoB (V194I), rrs (G878A), and pncA (Q122Stop) genes were observed. Ion Torrent sequencing may facilitate tracking and monitoring geographically diverse multidrug-resistant and extensively drug-resistant strains and could potentially be integrated into selected regional and reference settings throughout Africa, India, and China.

Vesiculation from Pseudomonas aeruginosa under SOS

Bacterial infections can be aggravated by antibiotic treatment that induces SOS response and vesiculation. This leads to a hypothesis concerning association of SOS with vesiculation. To test it, we conducted multiple analyses of outer membrane vesicles (OMVs) produced from the Pseudomonas aeruginosa wild type in which SOS is induced by ciprofloxacin and from the LexA noncleavable (lexAN) strain in which SOS is repressed. The levels of OMV proteins, lipids, and cytotoxicity increased for both the treated strains, demonstrating vesiculation stimulation by the antibiotic treatment. However, the further increase was suppressed in the lexAN strains, suggesting the SOS involvement. Obviously, the stimulated vesiculation is attributed by both SOS-related and unrelated factors. OMV subproteomic analysis was performed to examine these factors, which reflected the OMV-mediated cytotoxicity and the physiology of the vesiculating cells under treatment and SOS. Thus, SOS plays a role in the vesiculation stimulation that contributes to cytotoxicity.

Detection of botulinum toxin using an evanescent wave immunosensor

Using fluorescein isothiocyanate (FITC)-streptavidin, quartz fibre-immobilized antibody (FiAb) and the evanescent wave component of a light beam, detection of Botulinum Toxin-B (BoTX) is described. Exposure of 3-aminopropyltriethoxysilane/glutaraldehyde (APTS/GA) treated quartz fibres to increasing amounts of anti-BoTX Ab indicated toxin binding to increase in a linear fashion up to approximately 125 ng added Ab. Quantitation of bound BoTX and FiAb by Dot-Blot analysis using avidin-Horseradish peroxidase (HRP) conjugation indicated the presence of 0.27 and 0.67 pmoles, respectively. Inclusion of nonbiotinylated BoTX in sampling mixtures reduced fluorescence in a dose-dependent manner over a narrow concentration range (0-300 ng). Exposure of FiAb to a variety of venoms resulted in no reduction of BoTX binding suggesting detection of BoTX via immobilized anti-BoTX Ab to be very specific.

Effect of levels of acetate on the mevalonate pathway of Borrelia burgdorferi.

Borrelia burgdorferi, the agent of Lyme disease, is a spirochetal pathogen with limited metabolic capabilities that survives under highly disparate host-specific conditions. However, the borrelial genome encodes several proteins of the mevalonate pathway (MP) that utilizes acetyl-CoA as a substrate leading to intermediate metabolites critical for biogenesis of peptidoglycan and post-translational modifications of proteins. In this study, we analyzed the MP and contributions of acetate in modulation of adaptive responses in B. burgdorferi. Reverse-transcription PCR revealed that components of the MP are transcribed as individual open reading frames. Immunoblot analysis using monospecific sera confirmed synthesis of members of the MP in B. burgdorferi. The rate-limiting step of the MP is mediated by HMG-CoA reductase (HMGR) via conversion of HMG-CoA to mevalonate. Recombinant borrelial HMGR exhibited a Km value of 132 µM with a Vmax of 1.94 µmol NADPH oxidized minute−1 (mg protein)−1 and was inhibited by statins. Total protein lysates from two different infectious, clonal isolates of B. burgdorferi grown under conditions that mimicked fed-ticks (pH 6.8/37°C) exhibited increased levels of HMGR while other members of the MP were elevated under unfed-tick (pH 7.6/23°C) conditions. Increased extra-cellular acetate gave rise to elevated levels of MP proteins along with RpoS, CsrABb and their respective regulons responsible for mediating vertebrate host-specific adaptation. Both lactone and acid forms of two different statins inhibited growth of B. burgdorferi strain B31, while overexpression of HMGR was able to partially overcome that inhibition. In summary, these studies on MP and contributions of acetate to host-specific adaptation have helped identify potential metabolic targets that can be manipulated to reduce the incidence of Lyme disease.

Real-time RT-PCR assays for type and subtype detection of influenza A and B viruses

Influenza viruses type A (H3N2 and H1N1 subtypes) and B are the most prevalently circulating human influenza viruses. However, an increase in several confirmed cases of high pathogenic H5N1 in humans has raised concerns of a potential pandemic underscoring the need for rapid, point of contact detection. In this report, we describe development and evaluation of ‘type,’ i.e., influenza virus A and B, and ‘subtype,’ i.e., H1, H3, and H5, specific, single‐step/reaction vessel format, real‐time RT‐PCR assays using total RNA from archived reference strains, shell‐vial cultured and uncultured primary (throat swab/nasal wash) clinical samples. The type A and B specific assays detected all 16 influenza type A viruses and both currently circulating influenza B lineages (Yamagata and Victoria), respectively. ‘Type’ and ‘subtype’ specific assays utilize one common set of thermocycling conditions, are specific and highly sensitive (detection threshold of approximately 100 target template molecules). All clinical specimens and samples were evaluated using both the unconventional portable Ruggedized Advanced Pathogen Identification Device (RAPID) and standard laboratory bench LightCycler instruments. These potentially field‐deployable assays could offer significant utility for rapid, point of care screening needs arising from a pandemic influenza outbreak.