High Cell Specificity Research Articles

Application of bacteriophages for detection of foodborne pathogens

Bacterial contamination of food products presents a challenge for the food industry and poses a high risk for the consumer. Despite increasing awareness and improved hygiene measures, foodborne pathogens remain a threat for public health, and novel methods for detection of these organisms are needed. Bacteriophages represent ideal tools for diagnostic assays because of their high target cell specificity, inherent signal-amplifying properties, easy and inexpensive production, and robustness. Every stage of the phage lytic multiplication cycle, from the initial recognition of the host cell to the final lysis event, may be harnessed in several ways for the purpose of bacterial detection. Besides intact phage particles, phage-derived affinity molecules such as cell wall binding domains and receptor binding proteins can serve for this purpose. This review provides an overview of existing phage-based technologies for detection of foodborne pathogens, and highlights the most recent developments in this field, with particular emphasis on phage-based biosensors.

In vivo MRI cell tracking using perfluorocarbon probes and fluorine-19 detection.

This article presents a brief review of preclinical in vivo cell-tracking methods and applications using perfluorocarbon (PFC) probes and fluorine-19 ((19) F) MRI detection. Detection of the (19) F signal offers high cell specificity and quantification ability in spin density-weighted MR images. We discuss the compositions of matter, methods and applications of PFC-based cell tracking using ex vivo and in situ PFC labeling in preclinical studies of inflammation and cellular therapeutics. We also address the potential applicability of (19) F cell tracking to clinical trials.

Cell penetration and cell-selective drug delivery using α-helix peptides conjugated with gold nanoparticles

Cell penetrating peptides (CPPs) have been developed as vectors for molecular delivery into various cells for use in drug delivery, gene therapy and cancer treatment by their property transporting various molecules into cytoplasm. CPPs with high internalization, cell specificity, and low cytotoxicity have been considered to increase the applicability for cell engineering. Gold nanospheres (GNSs) are a useful tool for molecular imaging, because they are non-cytotoxic and have high solubility, ease of synthesis and excellent light scattering property. Here, we investigated the cell penetrability using α-helix peptides of 17-amino acids conjugated to gold nanospheres (P-GNS). Depending on the peptide sequence had the different cell penetrating (CP) activity for three kinds of cell lines. P-GNS showed low cytotoxicity and high selectivity against three cell types, despite just one amino acid difference between the peptide. We studied the cytotoxic activity of an anti-cancer drug doxorubicin (DOX) conjugated to the P-GNS. They showed different cytotoxicity against the three cell lines, depending on the peptide sequence, with a higher efficiency than free DOX at the same concentration. The cytotoxicity by DOX was correlated with the CP activity of the peptides against the three cell lines. These results demonstrated that P-GNS would be a useful tool for the development of a new cell-selective drug delivery system.

Comparison of progestin transcriptional profiles in rat mammary gland using Laser Capture Microdissection and whole tissue-sampling

Investigation of molecular mechanisms by gene expression profiling gains increasingly importance in preclinical safety evaluation. However, assigning expressed genes to specific cell populations is nearly impossible if the investigated RNA originates from whole tissue extracts. In this regard, Laser Capture Microdissection (LCM) can be used to detect changes specific to individual cell types. The objective of this study was to investigate the use of LCM for characterisation of progestin-related gene expression changes in the mammary gland. Thus, transcriptional profiles of the mammary gland of rats treated with a non-steroidal progesterone-receptor ligand, promegestone, medroxyprogesterone acetate, progesterone or vehicle were compared using whole tissue homogenates or LCM-captured epithelial cells. Total RNA from 30 mammary glands was isolated from snap-frozen specimen of the whole tissue and from approximately 25.000-30.000 cells of cresyl violet stained frozen sections employing LCM. After amplification of averaged 0.2μg total RNA of LCM-captured samples, RNA was labelled, hybridised to Affymetrix GeneChips and analysed. LCM-captured samples showed up to 3-fold more differentially expressed probe sets (progesterone) and up to 10-fold more downregulated (promegestone) probe sets than whole tissue samples implying high cell specificity. Moreover, mammary gland specific differentiation markers like whey acidic protein, alpha lactalbumin, casein alpha s1 and casein kappa showed up to 3.4-fold (alpha lactalbumin, vehicle) higher expression values. Multivariate data analyses revealed a clear separation of gene expression profiles according to the method used, suggesting an amplification dependent bias. LCM transcriptional profiling provides highly cell-specific information. An amplification dependent bias was observed. The technical variability was shown to be smaller than the biological variability. For progestin-related transcriptional profiling of the mammary gland, whole tissue-sampling proved to yield more informative results. Therefore LCM should only be considered when cell-type specific gene expression profiles are necessary for an in depth evaluation.

PACα- an optogenetic tool for in vivo manipulation of cellular cAMP levels, neurotransmitter release, and behavior in Caenorhabditis elegans

Photoactivated adenylyl cyclase α (PACα) was originally isolated from the flagellate Euglena gracilis. Following stimulation by blue light it causes a rapid increase in cAMP levels. In the present study, we expressed PACα in cholinergic neurons of Caenorhabditis elegans. Photoactivation led to a rise in swimming frequency, speed of locomotion, and a decrease in the number of backward locomotion episodes. The extent of the light-induced behavioral effects was dependent on the amount of PACα that was expressed. Furthermore, electrophysiological recordings from body wall muscle cells revealed an increase in miniature post-synaptic currents during light stimulation. We conclude that the observed effects were caused by cAMP synthesis because of photoactivation of pre-synaptic PACα which subsequently triggered acetylcholine release at the neuromuscular junction. Our results demonstrate that PACα can be used as an optogenetic tool in C. elegans for straightforward in vivo manipulation of intracellular cAMP levels by light, with good temporal control and high cell specificity. Thus, using PACα allows manipulation of neurotransmitter release and behavior by directly affecting intracellular signaling.

Generation of highly potent nonviral gene vectors by complexation of lipoplexes and transferrin-bearing fusogenic polymer-modified liposomes in aqueous glucose solution

We reported previously that complexation of lipoplexes containing 3,5-dipentadecyloxybenzamidine (TRX-20) and transferrin-bearing succinylated poly(glycidol) (SucPG)-modified liposome, which becomes fusogenic under weakly acidic conditions, might produce gene carriers with high transfection activity. For the present study, we prepared the lipoplex–SucPG-modified liposome complexes by mixing them either in phosphate-buffered saline or in an aqueous 5% glucose solution. The complexes prepared in phosphate-buffered saline have large particles of more than 800 nm, whereas the complexes prepared in the glucose solution were remarkably small: 200–300 nm. The small complexes were taken up more effectively by HeLa cells, and their transfection was induced more efficiently than the large complexes'. In addition, the small complexes achieved cellular transfection more efficiently in the presence of serum than in the absence of serum, without marked cytotoxicity. Considering that their affinity to the cell is based on ligand–receptor interaction, the small complexes are highly promising as a safe vector with high transfection activity and high target cell specificity.

Bacterial two-component and hetero-heptameric pore-forming cytolytic toxins: structures, pore-forming mechanism, and organization of the genes.

Staphylococcal gamma-hemolysin (Hlg), leukocidin (Luk), and Panton-Valentine leukocidin (PVL) are two-component and hetero-oligomeric pore-forming cytolytic toxins (or cytolysin), that were first identified in bacteria. No information on the existence of hetero-oligomeric pore-forming cytolytic toxins in bacteria except for staphylococcal strains is available so far. Hlg (Hlg1 of 34 kDa/Hlg2 of 32 kDa) effectively lyses erythrocytes from human and other mammalian species. Luk (LukF of 34 kDa/LukS of 33 kDa) is cytolytic toward human and rabbit polymorphonuclear leukocytes and rabbit erythrocytes, and PVL (LukF-PV of 34 kDa/LukS-PV of 33 kDa) reveals cytolytic activity with a high cell specificity to leukocytes. Hlg1 is identical to LukF and that the cell specificities of the cytolysins are determined by Hlg2 and LukS. Based on the primary and 3-dimensional structures of the toxin components, Hlg, Luk, and PVL are thought to form a family of proteins. In the first chapter of this article, we describe the molecular basis of the membrane pore-forming nature of Hlg, Luk, and PVL. We also describe a requirement of the phosphorylation of LukS and LukS-PV by protein kinase for their leukocytolytic activity besides their pore formation on human leukocytes.Recently, the assembly mechanism of the LukF and Hlg2 monomers into pore-forming hetero-oligomers of Hlg on human erythrocyte membranes has been clarified for the first time by our study using a single-molecular fluorescence imaging technique. We estimated 11 sequential equilibrium constants for the assembly pathway which includes the beginning with membrane binding of monomers, proceeds through single pore oligomerization, and culminates in the formation of clusters of the pores. In the second chapter of this article, we refer to an assembly mechanism of LukF and Hlg2 on human erythrocytes as well as the roles of the membranes of the target cells in pore formation by Hlg. The LukF, LukS, and Hlg2 proteins are derived from the Hlg locus (hlg), and have been found in 99% of clinical isolates of Staphylococcus aureus. In contrast, LukF-PV and LukS-PV are derived from the PVL locus (pvl) which is distinct from the hlg locus, and only a small percentage of clinically isolated S. aureus strains carries pvl. Recently, we discovered pvl on the genome of lysogenic bacteriophages, psiPVL, and determined the entire gene of the phage. We also demonstrated the phage conversion of S. aureus leading to the production of PVL through the discovery of a PVL-carrying temperate phage, psiSLT, from a clinical isolate of S. aureus. In the third chapter of this article, we discuss genetic analyses of the Hlg, Luk, and PVL genes. We also discuss the current status of knowledge of the genetic organization of PVL-converting phages in order to achieve an understanding of their molecular evolution.

Targeted Lysosome Disruptive Elements for Improvement of Parenchymal Liver Cell-specific Gene Delivery

The transfection ability of nonviral gene therapy vehicles is generally hampered by untimely lysosomal degradation of internalized DNA. In this study we describe the development of a targeted lysosome disruptive element to facilitate the escape of DNA from the lysosomal compartment, thus enhancing the transfection efficacy, in a cell-specific fashion. Two peptides (INF7 and JTS-1) were tested for their capacity to disrupt liposomes. In contrast to JTS-1, INF7 induced rapid cholesterol-independent leakage (EC(50), 1.3 microm). INF7 was therefore selected for coupling to a high affinity ligand for the asialoglycoprotein receptor (ASGPr), K(GalNAc)(2), to im- prove its uptake by parenchymal liver cells. Although the parent peptide disrupted both cholesterol-rich and -poor liposomes, the conjugate, INF7-K(GalNAc)(2), only induced leakage of cholesterol-poor liposomes. Given that endosomal membranes of eukaryotic cells contain <5% cholesterol, this implies that the conjugate will display a higher selectivity toward endosomal membranes. Although both INF7 and INF7-K(GalNAc)(2) were found to increase the transfection efficiency on polyplex-mediated gene transfer to parenchymal liver cells by 30-fold, only INF7-K(GalNAc)(2) appeared to do so in an ASGPr-specific manner. In mice, INF7-K(GalNAc)(2) was specifically targeted to the liver, whereas INF7 was distributed evenly over various organs. In summary, we have prepared a nontoxic cell-specific lysosome disruptive element that improves gene delivery to parenchymal liver cells via the ASGPr. Its high cell specificity and preference to lyse intracellular membranes make this conjugate a promising lead in hepatocyte-specific drug/gene delivery protocols.

Eotaxin Is a Potent Chemotaxin for Human Basophils

We studied the effect of eotaxin, a novel eosinophil-active CC chemokine with high target cell specificity, on human basophils. Eotaxin induced higher levels of chemotactic response with a lower ED50compared with RANTES in basophils; half-maximal migration occurred at a concentration of ∼3 nM. On the other hand, it exerted only a marginal effect on either histamine release or leukotriene C4generation. In addition, nested PCR amplification experiments revealed the expression of CC CKR3, a putative receptor for eotaxin, on basophils. Since accumulation of both basophils and eosinophils is an important aspect of allergic inflammation, eotaxin potentially plays a pathogenic role in allergic disorders by inducing migration of both of these cell types.

Production of Three Monoclonal Antibodies Specifically Reactive Respectively with the Ductal, Acinar and Islet Cells of the Human Pancreas

Three monoclonal antibodies, designated FP-1, FP-2 and FP-3, were obtained by immunizing a BALB/c mouse with dispersed human fetal pancreatic cells and using hybridoma technology. FP-1, FP-2 and FP-3 reacted specifically, respectively, with the ductal epithelial, acinar and islet cells, of the fetal pancreas as well as with adult human pancreatic tissue, suggesting a use in the discrimination of pancreatic cell types. Upon screening various tumor tissues, FP-1 was found to react with adenocarcinomas of the pancreas (16/23), stomach (7/8), colon (4/6) and gall bladder (2/2) as well as with the three malignant cell lines, PANC-1 (pancreas), HT-29 (colon) and LoVo (colon). In contrast to FP-1, FP-2 and FP-3 reacted with only 5% (2/39) and 0% (0/39), respectively, of these adenocarcinomas. Although FP-3 did not react with any of these adenocarcinomas, it did react with various APUDoma cells such as islet cell tumors of the pancreas (3/5), pheochromocytomas (2/2), medullary thyroid carcinomas (2/3) and gastrointestinal carcinoids (1/3). FP-3 thus appears to be the first monoclonal antibody with extremely high endocrine cell specificity.

Koch's postulates and the molecular biology of T-cell function

How can the function of a molecule on the surface of a T lymphocyte be determined? The advent of monoclonal antibodies (mAbs) and clonal populations of T cells seemed to make this task relatively simple: blocking of a particular function of a cloned T cell by a well- characterized mAb seems to provide ~easonable evi- dence for the involvement of the structure recognized by the mAb with the function that is blocked. For structures with demonstrable receptor function, adaptation of 'classical' approaches of endocrinology and cellular physiology assessing receptor-ligand interactions can provide confirmatory information. If a pure, reco,'qbinant lymphokine binds to a cell with characteristics appropri- ate for a hormone receptor (saturable binding with a reasonably high affinity, and ligand and target cell specificity), and if this binding is blocked by a mAb, it seems logical to designate the structure recognized by the antibody as the receptor for that lymphokine. However, defining functions of cell surface molecules by the inhibitory properties of mAbs is less satisfactory for complex structures and those for which a putative ligand has not been isolated in a form suitable for binding studies. The T-cell receptor (TCR) for antigen and several 'accessory' molecules found on T lymphocytes fit in this category. CD4 and CD8 are members of the immunoglobulin gene superfamily which are expressed on mutually exclusive subsets of mature peripheral T lymphocytes. The presence of these molecules on mature T cells generally correlates better with the class of major histocompatibility complex (MHC) antigen which restricts the reactivity of the cell than with its functional charac- teristics: CD4 is found on human and murine T cells which react with, or whose reactivity is restricted by, class II MHC antigens, while CD8 is found on human and murine T cells which react with, or whose reactivity is restricted by, class ! MHC antigens ~-3. This correlation, and the fact that mAbs reactive with CD4 or CD8 inhibited antigen-specific functions of T cells bearing these structures, suggested that CD4 and CD8 react with monomorphic determinants expressed on class II and I MHC molecules, respectively. However T cells vary in susceptibility to inhibition by anti-CD4 or anti-CD8 mAbs. For example, the cytotox- icity of most cloned cytolytic T lymphocytes (CTL) derived from primary mixed leukocyte cultures (MLC)is inhibited by anti-CD8 mAb, but this is not true for most cloned CTL derived from MLC prepared with spleen cells from mice sensitized in vivo with alloantigens 4.s. Cloned CIL less susceptible to inhibition by anti-CD8 mAb could also lyse target cells expressing low levels of specific allo- antigen, whereas cloned CTL highly susceptible to inhibi- tion by anti-CD8 mAb could not 6. Also, using a cloned CTL whicl~ interacted with two different target cells through the same TCR but with different avidities (based on cold target inhibition studies), the apparently lower The Ben May Institute, the Department of Pathology, and the Commit- tee on Immunology, The University of Chicago, Chicago, IL 60637, USA,

Neuron specific enolase in amniotic fluid--a possible indicator for fetal distress and brain implication.

It can be suggested that a NSE concentration in body fluids above the normal range may indicate brain damage, because NSE is found in neurons and neuroendocrine cells only. In this paper the concentration of the neuron specific enolase (NSE) in the amniotic fluid of normal and high risk pregnancies was investigated. Sixty-three samples of amniotic fluid were collected from 55 pregnant women and women in labor between the 19th and the 42nd gestational week. In 24 normal pregnancies 1.02 +/- 0.31 microgram NSE/L (mean +/- SD) were found. A relationship of NSE concentration was not found for gestational age, uterine contractions, age and parity of the mother, infant's birth weight or 1 min Apgar score. Maternal diseases such as diabetes mellitus, hypertension and others do not result in any increase of NSE concentration in the amniotic fluid (N = 20; 1.06 +/- 0.24 microgram NSE/L). Fetal and birth related high risk factors such as meconium stained amniotic fluid, cord around the neck, severe decelerations of the fetal heart rate or combinations of these factors caused the NSE concentration to exceed significantly normal values (N = 17; 2.22 +/- 0.87 microgram NSE/L). Due to its high organ and cell specificity, NSE may be a possible parameter for brain damage due to hypoxia in the fetus and newborn; however, only subsequent neurological and psychological examinations of these children can indicate the prognostic value of an elevation of NSE concentration in amniotic fluid.

Isolation of Actinomyces bacteriophage from human dental plaque.

Human dental plaque samples were screened for the presence of bacteriophage for Actinomyces viscosus and Streptococcus sanguis. None of the 336 samples yielded phage for S. sanguis, but 10 contained virulent actinomyces phage. A high host cell specificity was observed in that one phage isolate infected only A. viscosus T14V, eight phage isolates infected only A. viscosus MG-1, and one infected both strains. None was capable of productively infecting various other actinomyces strains that represented the six actinomyces coaggregation groups. Because phage-containing samples occurred randomly in this survey, no correlation between the individual collecting the samples, dental clinic, or type of patient and the presence of phage in the sample was noted. Examination of one of the samples that yielded phage for the presence of a natural host strain for that particular phage resulted in the isolation of two strains which were identified as A. viscosus serotype II and Actinomyces naeslundii serotype I. This is the first report of an A. naeslundii host strain and actinomyces bacteriophage of human dental plaque origin. The finding of both phage and host strains in the same dental plaque sample along with the observation of high host cell specificity by these phage provide indicators that support an active role for actinomyces bacteriophage in oral microbial ecology. The use of these freshly isolated phage as probes to study actinomyces coaggregation properties is discussed.