FlaC, a protein of Campylobacter jejuni TGH9011 (ATCC43431) secreted through the flagellar apparatus, binds epithelial cells and influences cell invasion.

Type III secretion systems identified in bacterial pathogens of animals and plants transpose effectors and toxins directly into the cytosol of host cells or into the extracellular milieu. Proteins of the type III secretion apparatus are conserved among diverse and distantly related bacteria. Many type III apparatus proteins have homologues in the flagellar export apparatus, supporting the notion that type III secretion systems evolved from the flagellar export apparatus. No type III secretion apparatus genes have been found in the complete genomic sequence of Campylobacter jejuni NCTC11168. In this study, we report the characterization of a protein designated FlaC of C. jejuni TGH9011. FlaC is homologous to the N- and C-terminus of the C. jejuni flagellin proteins, FlaA and FlaB, but lacks the central portion of these proteins. flaC null mutants form a morphologically normal flagellum and are highly motile. In wild-type C. jejuni cultures, FlaC is found predominantly in the extracellular milieu as a secreted protein. Null mutants of the flagellar basal rod gene (flgF) and hook gene (flgE) do not secrete FlaC, suggesting that a functional flagellar export apparatus is required for FlaC secretion. During C. jejuni infection in vitro, secreted FlaC and purified recombinant FlaC bind to HEp-2 cells. Invasion of HEp-2 cells by flaC null mutants was reduced to a level of 14% compared with wild type, suggesting that FlaC plays an important role in cell invasion.

Functional classification of interferon-stimulated genes identified using microarrays.

Interferons (IFNs) are a family of multifunctional cytokines that activate transcription of subsets of genes. The gene products induced by IFNs are responsible for IFN antiviral, antiproliferative, and immunomodulatory properties. To obtain a more comprehensive list and a better understanding of the genes regulated by IFNs, we compiled data from many experiments, using two different microarray formats. The combined data sets identified >300 IFN-stimulated genes (ISGs). To provide new insight into IFN-induced cellular phenotypes, we assigned these ISGs to functional categories. The data are accessible on the World Wide Web at http://www.lerner.ccf.org/labs/williams/, including functional categories and individual genes listed in a searchable database. The entries are linked to GenBank and Unigene sequence information and other resources. The goal is to eventually compile a comprehensive list of all ISGs. Recognition of the functions of the ISGs and their specific roles in the biological effects of IFNs is leading to a greater appreciation of the many facets of these intriguing and essential cytokines. This review focuses on the functions of the ISGs identified by analyzing the microarray data and focuses particularly on new insights into the protein kinase RNA-regulated (PRKR) protein, which have been made possible with the availability of PRKR-null mice.

Model-based target recognition in pulsed ladar imagery.

A pulsed ladar based object-recognition system with applications to automatic target recognition (ATR) is presented. The approach used is to fit the sensed range images to range templates extracted through a laser physics based simulation applied to geometric target models. A projection-based prescreener filters out more than 80% of candidate templates. For recognition, an M of N pixel matching scheme for internal shape matching is combined with a silhouette matching scheme. The system was trained on synthetic data obtained from the simulation, and has been blind tested on a data set containing real ladar images of military vehicles at various orientations and ranges. Successful blind testing on real imagery demonstrates the utility of synthetic imagery for training of recognizers operating on ladar imagery.

Effect of deficiency of the double-stranded RNA-dependent protein kinase, PKR, on antiviral resistance in the presence or absence of ribonuclease L: HSV-1 replication is particularly sensitive to deficiency of the major IFN-mediated enzymes.

Control of viral replication by interferon (IFN) is thought to be principally mediated by the 2',5'-oligoadenylate synthetase (OAS)/RNAse L, double-stranded dependent protein kinase (PKR), and myxovirus resistance protein (Mx) pathways. In this study, we monitored the constitutive and IFN-induced antiviral activity in mouse embryo fibroblasts lines derived from mice with targeted disruption of either PKR or PKR/RNAse L genes. At high multiplicity of infection (moi = 10), the absence of PKR had no effect on replication of vesicular stomatitis virus (VSV) but moderately enhanced encephalomyocarditis virus (EMCV) growth and greatly increased replication of herpes simplex virus-1 (HSV-1). Replication of EMCV, HSV-1, and VSV was modestly higher in PKR-/- RNAse L-/- fibroblasts when compared with control cells. Although the antiviral action of IFN-alpha was unaffected by the absence of PKR, IFN action was significantly impaired in the double knockout cells but was dependent on the stage of the virus cycle. At early stages, it appeared that anti-EMCV and anti-HSV-1 action of IFN-alpha was significantly compromised, although weak residual antiviral activity was seen. The action of IFN-alpha against VSV was specifically compromised at a late stage of virus replication. The results showed that PKR is an important mediator in constitutive resistance against HSV-1 and that RNAse L is also necessary for the full antiviral activity of IFN against a variety of viruses. These results supported the existence of novel pathways aimed toward specific stages of the virus life cycle.

Central role of double-stranded RNA-activated protein kinase in microbial induction of nitric oxide synthase.

NO synthase 2 (NOS2) is induced in airway epithelium by influenza virus infection. NOS2 induction late in the course of viral infection may occur in response to IFN-gamma, but early in infection gene expression may be induced by the viral replicative intermediate dsRNA through the dsRNA-activated protein kinase (PKR). Since PKR activates signaling pathways important in NOS2 gene induction, we determined whether PKR is a component in the signal transduction pathway leading to NOS2 gene expression after viral infection of airway epithelium. We show that NOS2 gene expression in human airway epithelial cells occurs in response to influenza A virus or synthetic dsRNA. Furthermore, dsRNA leads to rapid activation of PKR, followed by activation of signaling components including NF-kappaB and IFN regulatory factor 1. NOS2 expression is markedly diminished and IFN regulatory factor 1 and NF-kappaB activation are substantially impaired in PKR null cells. Strikingly, NOS2 induction in response to LPS is abolished in PKR null cells, confirming a central role for PKR in the general signaling pathway to NOS2.

Content of non-mercury-associated selenium in human tissues.

Recent studies have shown that at a higher mercury (Hg) burden, the molar ratio of selenium (Se) and Hg in tissues tends to approximate 1:1 by the formation of biologically largely inert adducts. From the toxicological standpoint, this trapping of free Hg is welcome. However, this binding of Se to Hg reduces the portion of Se in tissues, which is available for the formation of essential selenoenzymes like glutathione peroxidase, type I deiodase, and so forth and could result in a relative deficiency of Se. Therefore, we tried to determine the concentration of non-Hg-associated Se in several human tissues. As there is no proved trace method for the speciation of non-Hg-bound and Hg-bound Se in tissues, the total concentrations of Hg and Se were determined and the portion of non-Hg-associated Se was calculated by the difference of the molar concentrations of Se and Hg. For this investigation, the following tissues were obtained by autopsy from 133 adults: kidney cortex, thyroid gland, liver, spleen, cerebrum cortex, and pituitary gland. In no case was an occupational Hg burden known. The results confirm the assumption of a 1:1 association of Hg and Se in human tissues. The mean concentration of non-Hg-bound Se was calculated to 576 microg/kg in the kidney cortex, 363 microg/kg in the thyroid gland, 308 microg/kg in the liver, 205 microg/kg in the spleen, 111 microg/kg in the cerebrum cortex, and 545 microg/kg in the pituitary gland. In none of the cases under investigation in any tissue was the molar Se/Hg ratio below 1. This means that a total deficiency of non-Hg-bound Se could not be seen in this normal population, even at a higher Hg burden. Nevertheless, at a suboptimal Se supply like in Germany, any reduction of the part of Se, which is available for the formation of essential seleno-enzymes, should be avoided. Therefore, any additional Hg burden such as from dental amalgam should to be considered critically. The different distribution of Hg and Se in the body confirms that there is a controlled hierarchy in the Se supply of different organs, which tries to prevent a Se deficiency in organs with essential seleno-enzymes like the thyroid gland even under an suboptimal Se supply.

Interferon action in triply deficient mice reveals the existence of alternative antiviral pathways.

Antiviral proteins encoded by the interferon (IFN)-stimulated genes provide a front-line defense against viral infections. In particular, PKR, RNase L, and Mx are considered to be the principal proteins through which IFNs mount an antiviral state. To determine whether alternative antiviral pathways exist, RNase L-/- mice and PKR-/- mice were crossed onto an Mx1(-/-) background to generate a strain of triply deficient (TD) mice. After infections with encephalomyocarditis virus, the TD mice died 3-4 days earlier than infected, wild-type mice. However, there was an IFN dose-dependent increase in survival times after encephalomyocarditis virus infections for both the TD and wild-type mice. Mice that were deficient for PKR or RNase L showed intermediate survival times between those of the TD and wild-type mice. Surprisingly, cultured embryonic fibroblasts lacking RNase L, PKR, or both proteins were still able to mount a substantial residual antiviral response against encephalomyocarditis virus or vesicular stomatitis virus after IFN-alpha treatments. These results confirm the antiviral functions of RNase L and PKR in vivo but also provide unequivocal evidence for the existence of novel, innate immune pathways against viruses.

Inhibition of double-stranded RNA- and tumor necrosis factor alpha-mediated apoptosis by tetratricopeptide repeat protein and cochaperone P58(IPK).

P58(IPK) is a tetratricopeptide repeat-containing cochaperone that is involved in stress-activated cellular pathways and that inhibits the activity of protein kinase PKR, a primary mediator of the antiviral and antiproliferative properties of interferon. To gain better insight into the molecular actions of P58(IPK), we generated NIH 3T3 cell lines expressing either wild-type P58(IPK) or a P58(IPK) deletion mutant, DeltaTPR6, that does not bind to or inhibit PKR. When treated with double-stranded RNA (dsRNA), DeltaTPR6-expressing cells exhibited a significant increase in eukaryotic initiation factor 2alpha phosphorylation and NF-kappaB activation, indicating a functional PKR. In contrast, both of these PKR-dependent events were blocked by the overexpression of wild-type P58(IPK). In addition, the P58(IPK) cell line, but not the DeltaTPR6 cell line, was resistant to dsRNA-induced apoptosis. Together, these findings demonstrate that P58(IPK) regulates dsRNA signaling pathways by inhibiting multiple PKR-dependent functions. In contrast, both the P58(IPK) and DeltaTPR6 cell lines were resistant to tumor necrosis factor alpha-induced apoptosis, suggesting that P58(IPK) may function as a more general suppressor of programmed cell death independently of its PKR-inhibitory properties. In accordance with this hypothesis, although PKR remained active in DeltaTPR6-expressing cells, the DeltaTPR6 cell line displayed a transformed phenotype and was tumorigenic in nude mice. Thus, the antiapoptotic function of P58(IPK) may be an important factor in its ability to malignantly transform cells.

Cell cycle regulation of the double stranded RNA activated protein kinase, PKR.

The interferon (IFN)-induced, double stranded RNA (dsRNA)-activated serine/threonine kinase, PKR, is a potent negative regulator of cell growth when overexpressed in yeast or mammalian cells. To determine whether endogenous PKR plays a role in cell growth control, we have investigated the regulation of PKR levels and activity during the cell cycle in human glioblastoma T98G cells. The steady-state level of PKR mRNA in T98G cells was highest in growth arrested cells, dropped sharply within 3 h of serum stimulation then gradually increased as cells progressed through G1, reaching a plateau in early S phase. PKR protein level increased following serum stimulation reaching a peak at the G2+M boundary and declining thereafter. In contrast, PKR kinase activity exhibited two peaks, in early G1 and at the G1/S boundary, declining sharply in early S phase. Thus, the activity profile did not follow the protein profile indicating a tight regulation of PKR at the level of activity. In T98G cells expressing the catalytically inactive PKRK296R the dsRNA-induced activation of NF-kappaB and IRF-1 was suppressed and the mutant cells exhibited resistance to stress induced apoptosis. Cell cycle distribution analysis showed that the mutant expressing cells exhibited longer G1 phase and fewer cells engaged in S phase. Furthermore, early passage mouse embryo fibroblasts derived from PKR knockout mice grew more slowly compared with the control cells. Taken together these results suggest that PKR may play a role in cell cycle progression.

Identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays.

The pleiotropic activities of interferons (IFNs) are mediated primarily through the transcriptional regulation of many downstream effector genes. The mRNA profiles from IFN-alpha, -beta, or -gamma treatments of the human fibrosarcoma cell line, HT1080, were determined by using oligonucleotide arrays with probe sets corresponding to more than 6,800 human genes. Among these were transcripts for known IFN-stimulated genes (ISGs), the expression of which were consistent with previous studies in which the particular ISG was characterized as responsive to either Type I (alpha, beta) or Type II (gamma) IFNs, or both. Importantly, many novel IFN-stimulated genes were identified that were diverse in their known biological functions. For instance, several novel ISGs were identified that are implicated in apoptosis (including RAP46/Bag-1, phospholipid scramblase, and hypoxia inducible factor-1alpha). Furthermore, several IFN-repressed genes also were identified. These results demonstrate the usefulness of oligonucleotide arrays in monitoring mammalian gene expression on a broad and unprecedented scale. In particular, these findings provide insights into the basic mechanisms of IFN actions and ultimately may contribute to better therapeutic uses for IFNs.

Automatic target recognition using a feature-decomposition and data-decomposition modular neural network.

A modular neural network classifier has been applied to the problem of automatic target recognition using forward-looking infrared (FLIR) imagery. The classifier consists of several independently trained neural networks. Each neural network makes a decision based on local features extracted from a specific portion of a target image. The classification decisions of the individual networks are combined to determine the final classification. Experiments show that decomposition of the input features results in performance superior to a fully connected network in terms of both network complexity and probability of classification. Performance of the classifier is further improved by the use of multiresolution features and by the introduction of a higher level neural network on the top of the individual networks, a method known as stacked generalization. In addition to feature decomposition, we implemented a data-decomposition classifier network and demonstrated improved performance. Experimental results are reported on a large set of real FLIR images.

A double-stranded RNA-activated protein kinase-dependent pathway mediating stress-induced apoptosis.

Apoptosis occurs in response to different cellular stresses, including viral infection, inflammatory cytokines, growth factor deprivation, and UV light, but it is unclear whether these inducers share a common mechanism of induction. The interferon-induced, double-stranded RNA-activated protein kinase (PKR) has been implicated in processes that rely on apoptosis as control mechanisms in vivo, including antiviral activities, cell growth regulation, and tumorigenesis. Here we report that mouse embryo fibroblasts from mutant mice containing homozygous deletions in the PKR gene (Pkr(0/0) mice) were resistant to apoptotic cell death in response to double-stranded RNA, tumor necrosis factor-alpha, or lipopolysaccharide. The mechanism underlying the suppression of apoptosis in the Pkr(0/0) cells could be attributed to defects in the activation of DNA-binding activity for the transcription factor interferon regulatory factor-1 and in Fas mRNA induction. Thus, these results provide genetic evidence implicating a requirement for PKR in mediating different forms of stress-related apoptosis.

Deficient cytokine signaling in mouse embryo fibroblasts with a targeted deletion in the PKR gene: role of IRF-1 and NF-kappaB.

The interferon (IFN)-induced double-stranded RNA (dsRNA)-activated Ser/Thr protein kinase (PKR) plays a role in the antiviral and antiproliferative effects of IFN. PKR phosphorylates initiation factor eIF2alpha, thereby inhibiting protein synthesis, and also activates the transcription factor, nuclear factor-kappaB (NF-kappaB), by phosphorylating the inhibitor of NF-kappaB, IkappaB. Mice devoid of functional PKR (Pkr(o/o)) derived by targeted gene disruption exhibit a diminished response to IFN-gamma and poly(rI:rC) (pIC). In embryo fibroblasts derived from Pkr(o/o) mice, interferon regulatory factor 1 (IRF-1) or guanylate binding protein (Gbp) promoter-reporter constructs were unresponsive to IFN-gamma or pIC but response could be restored by co-transfection with PKR. The lack of responsiveness could be attributed to a diminished activation of IRF-1 and/or NF-kappaB in response to IFN-gamma or pIC. Thus, PKR acts as a signal transducer for IFN-stimulated genes dependent on the transcription factors IRF-1 and NF-kappaB.

Probe-based automatic target recognition in infrared imagery.

A probe-based approach combined with image modeling is used to recognize targets in spatially resolved, single frame, forward looking infrared (FLIR) imagery. A probe is a simple mathematical function that operates locally on pixel values and produces an output that is directly usable by an algorithm. An empirical probability density function of the probe values is obtained from a local region of the image and used to estimate the probability that a target of known shape is present. Target shape information is obtained from three-dimensional (3-D) computer-aided design (CAD) models. Knowledge of the probe values along with probe probability density functions and target shape information allows the likelihood ratio between a target hypothesis and background hypothesis to be written. The generalized likelihood ratio test is then used to accept one of the target poses or to choose the background hypothesis. We present an image model for infrared images, the resulting recognition algorithm, and experimental results on three sets of real and synthetic FLIR imagery.

Simulation of error in optical radar range measurements.

We describe a computer simulation of atmospheric and target effects on the accuracy of range measurements using pulsed laser radars with p-i-n or avalanche photodiodes for direct detection. The computer simulation produces simulated images as a function of a wide variety of atmospheric, target, and sensor parameters for laser radars with range accuracies smaller than the pulse width. The simulation allows arbitrary target geometries and simulates speckle, turbulence, and near-field and far-field effects. We compare simulation results with actual range error data collected in field tests.

Involvement of the double-stranded-RNA-dependent kinase PKR in interferon expression and interferon-mediated antiviral activity.

The signaling mechanisms responsible for the induced expression of interferon (IFN) genes by viral infection or double-stranded RNA (dsRNA) are not well understood. Here we investigate the role of the interferon-induced dsRNA-dependent protein kinase PKR in the regulation of IFN induction. Biological activities attributed to PKR include regulating protein synthesis, mediating IFN actions, and functioning as a possible tumor suppressor. Since binding of dsRNA is required for its activation, PKR has been considered as a candidate signal transducer for regulating IFN expression. To examine this role of PKR, loss-of-function phenotypes in stable transformants of promonocytic U-937 cells were achieved by two different strategies, overexpression of an antisense PKR transcript or a dominant negative PKR mutant gene. Both types of PKR-deficient cells were more permissive for viral replication than the control U-937 cells. As the result of PKR loss, they also showed impaired induction of IFN-alpha and IFN-beta genes in response to several inducers--specifically, encephalomyocarditis virus, lipopolysaccharide, and phorbol 12-myristate 13-acetate. Interestingly, while IFN-alpha induction by dsRNA was impaired in PKR-deficient cells, IFN-beta induction remained intact. Loss of PKR function also resulted in decreased antiviral activity as elicited by IFN-alpha and, to a greater extent, by IFN-gamma. These results implicate PKR in the regulation of several antiviral activities.

Regulation of tumor necrosis factor receptor expression by acid-labile interferon-alpha from AIDS sera.

High levels of an unusual acid-labile interferon (IFN) alpha in sera of patients with human immunodeficiency virus (HIV) infection are associated with disease progression to acquired immunodeficiency syndrome (AIDS). Since IFNs have been shown to enhance the cytotoxic actions of tumor necrosis factor (TNF), a potent mediator of inflammation and cachexia, a study was undertaken to investigate whether the acid-labile IFN alpha produced in AIDS can regulate TNF receptor expression. The expression of TNF receptors was determined by studying the interaction of [125I]TNF with cellular receptors. The results show the acid-labile IFN alpha present in AIDS sera is capable of inducing the expression of cellular receptors for TNF. The extent of induction of TNF receptors depends on the concentration of the acid-labile IFN alpha in the AIDS sera. There is no significant induction of TNF receptors when the AIDS sera are preneutralized with polyclonal anti-IFN alpha antibodies. It is also shown that the synthesis of TNF by peripheral blood monocytes (PBM) from patients with HIV infection is enhanced during the progression of HIV infection in vivo. Thus, the TNF system is activated in patients with HIV infection. This activation may be a contributing factor to some of the physiological disturbances including the wasting syndrome observed in AIDS.

Fluorescence assay to monitor phagocytosis by bloodclot derived polymorphonuclear leucocytes. 2. Study of patients with psoriasis and the effect of exposing leucocytes to psoriatic skin scales.

Comparisons of phagocytic parameters were carried out by a recently developed fluorescence test which is reproducible, simple and fast. Phagocytosis by polymorphonuclear leucocytes (PMNs) obtained from patients with psoriasis was compared with that of healthy individuals. Psoriatic skin scales, non-sterile and sterile, were tested for stimulatory effect on PMNs and compared with the effect of normal skin scrapings. Results confirm enhanced phagocytosis of bacteria by PMNs from patients with psoriasis over that of PMNs from healthy volunteers. Furthermore, the supernatant fluid from suspensions of psoriatic skin scales, non-sterile and sterile, stimulated PMNs activity.

The effect of 5-fluorouracil on the mitotic cycle and DNA synthesis in the epithelium of mouse small intestine.

The examinations were performed on 42 mice of the Porton strain. The experimental animals were injected intraperitoneally with the dose of 75 mg of 5-fluorouracil per kg body weight. The first experimental group received injections of [3H]thymidine within 48 hours and the second group within 96 hours of the injection of 5-fluorouracil. Two mice from each group were killed at within 1, 2, 4, 8, 12, 24, and 48 hours of the [3H]thymidine injection. Calculations of the mitotic index and time of duration of individual phases of the mitotic cycle in epithelial cells of the small intestine were based on application of the autoradiographic method. These studies lead to the conclusion that 5-fluorouracil disturbs the course of metabolic processes in the cell, which are also related with the distribution of the genetic material. Histological examinations show that 5-fluorouracil produces profound morphological changes in the intestine, which affect both the intestinal epithelium and the connective tissue stroma. The autoradiographic tests revealed a considerable suppression of the mitotic activity of the epithelium of intestinal crypts. Moreover, it was shown that 5-fluorouracil inhibits the mitotic activity of the intestinal epithelium by diminishing the number of cells capable of entering into mitosis. Nevertheless, by 96 hours following introduction of a single dose of 5-fluorouracil normal morphological structure and mitotic activity of the intestinal wall cells are restored.