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P Luo

Publications and source records attributed to P Luo.

At least 19 recordsLinked to original sources
PubMed2006

A self-augmenting gene expression cassette for enhanced and sustained transgene expression in the presence of proinflammatory cytokines.

Viral promoters can yield high gene expression levels yet tend to be attenuated in vivo by host proinflammatory cytokines. Prolonged transgene expression can be obtained using constitutive cellular promoters. However, levels of transgene expression driven by cellular promoters are insufficient for effective therapy. We designed a novel self-augmenting gene expression cassette in which the transgene product can induce an endogenous transcription factor to enhance the activity of a weak cellular promoter driving its expression. Using the cellular major histocompatibility complex class I (H-2K(b)) promoter to drive the interferon (IFN-gamma) cytokine gene, we show that the H-2K(b) promoter, although exhibiting much lower basal activity, yields higher IFN-gamma production than the CMV promoter 2 days after transfection. IFN-gamma expression driven by the H-2K(b) promoter also lasts longer than that driven by the cytomegalovirus promoter. Our data demonstrate that the self-augmenting strategy provides a promising approach to achieve high and sustained transgene expression in vivo.

Animals↗
PubMed2005

Monosynaptic circuitry of trigeminal proprioceptive afferents coordinating jaw movement with visceral and laryngeal activities in rats.

Jaw movement is intimately related to various oromotor and visceral functions such as feeding, swallowing, vocalization, respiration and cardiac function. Neuronal circuitry that links jaw movement and these visceral and oromotor functions is largely unknown. The purpose of this study is to determine whether the trigeminal proprioceptive and jaw-muscle spindle afferents send axons to and synapse with the motoneurons innervating visceral organs and laryngeal muscles utilizing multiple double-labeling and physiological approaches. Double labeling of anterograde tracing combined to retrograde transport was performed by injection of biotinylated dextran amine into the mesencephalic trigeminal nucleus and cholera toxin B subunit or horseradish peroxidase into the vagus nerve and the recurrent laryngeal nerve. Mesencephalic trigeminal nucleus neuronal terminals contacted with visceral and laryngeal muscle motoneurons in the ambiguus nucleus and the nearby intermediate reticular zone. By electron microscopic observation, we confirmed that mesencephalic trigeminal nucleus terminals made asymmetric axodendritic synapses with these motoneurons. Double labeling of physiologically characterized jaw muscle spindle afferent neurons combined with anti-choline acetyltransferase immunohistochemistry showed that jaw-muscle spindle afferent boutons closely contacted choline acetyltransferase-immunoreactive motoneurons in the ambiguus nucleus and intermediate reticular zone. This report is the first to demonstrate that the trigeminal proprioceptive afferents synapsed upon visceral and laryngeal muscle motoneurons and provide neuronal networks for the jaw-visceral and jaw-laryngeal coordination.

Afferent Pathways↗
PubMed2005

Novel amplifier expression vectors producing higher levels of IL-2 led to slower tumor growth and longer survival in vivo.

Sufficient levels of gene expression are required for effective gene therapy. One of the major obstacles in gene therapy is the low transgene expression obtained from currently available vector systems. To address this issue, we employed a transcriptional amplifier strategy in a single construct to enhance transgene expression. In the amplifier vectors (pHi-1 and pHi-2), the strong CMV promoter was used to drive a transcriptional factor, Tat, which could transactivate a second promoter (HIV1 LTR or HIV2 LTR) located in the same construct driving the gene of interest. Using the human interleukin-2 (IL-2) cytokine gene, our data showed that the pHi-1/2 amplifier vectors could produce significantly higher IL-2 levels in human lung cancer cells (A549) and breast cancer cells (MCF-7) than that obtained by directly using the CMV promoter alone. Injection of pHi-2-IL-2-modified Lewis Lung (LL/2) tumor clones led to significantly slower tumor growth and longer survival in mice compared to those injected with either CMV promoter driven IL-2 clones or the parental tumor cells. Our results demonstrated that the transcriptional amplifier-based expression cassettes could be very useful in applications where high levels of gene expression are difficult to achieve.

Animals↗
PubMed2003

Fucosylated lactosamines participate in adhesion of HIV-1 envelope glycoprotein to dendritic cells.

Carbohydrates expressed on HIV-1 gp160 are purported to bind to several receptor types that affect virus pathophysiology. Here, we define a potential role for fucosylated glycans involved in the adhesion of cells expressing anchored HIV-1 glycoprotein or HIV virions to human dendritic cells (DCs). We observe that a monoclonal antibody (FH6), with reactivity toward an extended dimeric form of a fucosyl lactosamine, binds to gp120 transfectants, blocking adhesion of these cells and virus particles to human DCs. We observe that serum antibodies induced by peptide mimetic of fucosylated carbohydrate core structures emulate the monoclonal antibody reactivity pattern, showing enhanced reactivity to HIV-1 envelope-expressing cell line and blocking the adhesion of these cells to human DCs. These results suggest a potential role for initial adherence of virally infected cells or virions mediated by fucosylated lactosamines expressed on the envelope protein. As these carbohydrates function as adhesion molecules associated with homing and dissemination processes, such interactions may contribute to the HIV infection process.

Animals↗
PubMed2003

Peptide mimotopes as prototypic templates of broad-spectrum surrogates of carbohydrate antigens.

Peptide mimetics of carbohydrate antigens can function as templates to exploit immune mechanisms to augment vaccine design strategies as they are T cell dependent antigens. In this study we evaluate a peptide mimetic (peptide 105) of the Pneumococcal capsular polysaccharide type 14 (Pn14) as a model antigen to explore differences in antigenicity and immunogenicity of peptide mimotopes. The multiple antigenic peptide (MAP) form, by ELISA, competes with native Pn14 in a concentration-dependent manner for binding to an anti-Pn14 monoclonal antibody. It was observed that peptide priming with a conjugated form (105-BSA) and boosting with Pn14 produced higher levels of Pn14-reactive IgG1, IgG2a, IgG2b and IgG3 than priming and boosting with Pn14. This serum also displayed reactivity with multiple serotypes, as assessed by ELISA. However, when compared with serum from humans immunized with the 23-valent pneumococcal vaccines, mimetic-induced mouse serum did not display a significant ability to mediate opsonophagocytic killing of pneumococci. These results suggest the feasibility of designing mimotopes to render effective humoral responses not only to a single serotype of Streptococcus pneumoniae, but to multiple serotypes at once. Such peptides would simplify currently available vaccine approaches, yet highlights the requirement of more extensive polymerization to fully emulate native antigen.

Antibodies, Monoclonal↗
PubMed2001

Cavity approach to noisy learning in nonlinear perceptrons.

We analyze the learning of noisy teacher-generated examples by nonlinear and differentiable student perceptrons using the cavity method. The generic activation of an example is a function of the cavity activation of the example, which is its activation in the perceptron that learns without the example. Mean-field equations for the macroscopic parameters and the stability condition yield results consistent with the replica method. When a single value of the cavity activation maps to multiple values of the generic activation, there is a competition in learning strategy between preferentially learning an example and sacrificing it in favor of the background adjustment. We find parameter regimes in which examples are learned preferentially or sacrificially, leading to a gap in the activation distribution. Full phase diagrams of this complex system are presented, and the theory predicts the existence of a phase transition from poor to good generalization states in the system. Simulation results confirm the theoretical predictions.

Biophysical Phenomena↗
PubMed2001

Light and electron microscopic observations of a direct projection from mesencephalic trigeminal nucleus neurons to hypoglossal motoneurons in the rat.

A direct projection from rat mesencephalic trigeminal nucleus (Vme) neurons to the hypoglossal nucleus (XII) motoneurons was studied using a double labeling method of anterogradely biotinylated dextran amine (BDA) tracing combined with retrogradely horseradish peroxidase (HRP) transport at both light and electron microscopic levels. BDA was iontophoresed unilaterally into the caudal Vme, and 7 days later HRP was injected into the ipsilateral tongue to label hypoglossal motoneurons. The BDA-labeled fibers were seen descended along Probst' tract and were traced to the caudal medulla. In this course, the fibers gave off axon collaterals bearing varicosities in the trigeminal motor nucleus (Vmo), the parvicellular reticular formation (PCRt), the dorsomedial portions of the subnuclei of oralis (Vodm) and interpolaris (Vidm) and in the XII ipsilaterally. The labeling of terminals was most dense in the PCRt at the levels of caudal pons and rostral medulla, which displayed a "dumbbell-shaped" form in the transverse planes. In the XII, labeled terminals were distributed mainly in the dorsal compartment of the nucleus. One hundred sixty-eight appositions made by BDA-labeled terminals on HRP-labeled motoneurons were seen in the dorsal compartment (71%) and in the lateral subcompartment (24%) of the ventral XII. Under electron microscopy BDA-labeled boutons containing clear, spherical synaptic vesicles were found to form synaptic contacts with the somata and dendrites of hypoglossal motoneurons with asymmetric specializations. The present study provides new evidence that the trigeminal proprioceptive afferent neurons terminate in the XII and make synaptic contacts with their motoneurons.

Animals↗
PubMed2001

Jaw-muscle spindle afferent pathways to the trigeminal motor nucleus in the rat.

Neural pathways conveying proprioceptive feedback from the jaw muscles were studied in rats by combining retrograde and intracellular neuronal labeling. Initially, horseradish peroxidase was iontophoresed unilaterally into the trigeminal motor nucleus (Vmo). Two days later, 1-5 jaw-muscle spindle afferent axons located in the mesencephalic trigeminal nucleus were physiologically identified and intracellularly stained with biotinamide. Stained mesencephalic trigeminal jaw-muscle spindle afferent axon collaterals and boutons were predominantly distributed in the supratrigeminal region (Vsup), Vmo, dorsomedial trigeminal principal sensory nucleus (Vpdm), parvicellular reticular formation (PCRt), alpha division of the parvicellular reticular formation (PCRtA), and dorsomedial portions of the spinal trigeminal subnuclei oralis (Vodm), and interpolaris (Vidm). Numerous neurons retrogradely labeled with horseradish peroxidase from the trigeminal motor nucleus were found bilaterally in the PCRt, PCRtA, Vodm, and Vidm. Retrogradely labeled neurons were also present contralaterally in the Vsup, Vpdm, Vmo, peritrigeminal zone, and bilaterally in the dorsal medullary reticular field. Putative contacts between intracellularly stained mesencephalic trigeminal jaw-muscle spindle afferent boutons and trigeminal premotor neurons retrogradely labeled with horseradish peroxidase were found in the ipsilateral Vodm, PCRtA, and PCRt, as well as the contralateral Vsup, Vmo, Vodm, PCRt, and PCRtA. Thus, multiple disynaptic jaw-muscle spindle afferent-motoneuron circuits exist. These pathways are likely to convey long-latency jaw-muscle stretch reflexes and may contribute to stiffness regulation of the masticatory muscles.

Afferent Pathways↗
PubMed2001

Directing the immune response to carbohydrate antigens.

Peptide mimetics may substitute for carbohydrate antigens in vaccine design applications. At present, the structural and immunological aspects of antigenic mimicry, which translate into immunologic mimicry, as well as the functional correlates of each, are unknown. In contrast to screening peptide display libraries, we demonstrate the feasibility of a structure-assisted vaccine design approach to identify functional mimeotopes. By using concanavalin A (ConA), as a recognition template, peptide mimetics reactive with ConA were identified. Designed peptides were observed to compete with synthetic carbohydrate probes for ConA binding, as demonstrated by enzyme-linked immunosorbent assay and isothermal titration calorimetry (ITC) analysis. ITC measurements indicate that a multivalent form of one particular mimetic binds to ConA with similar affinity as does trimannoside. Splenocytes from mimeotope-immunized mice display a peptide-specific cellular response, confirming a T-cell-dependent nature for the mimetic. As ConA binds to the Envelope protein of the human immunodeficiency virus, type 1 (HIV-1), we observed that mimeotope-induced serum also binds to HIV-1-infected cells, as assessed by flow cytometry, and could neutralize T-cell line adapted HIV-1 isolates in vitro, albeit at low titers. These studies emphasize that mimicry is based more upon functional rather than structural determinants that regulate mimeotope-induced T-dependent antibody responses to polysaccharide and emphasize that rational approaches can be employed to develop further vaccine candidates.

Amino Acid Sequence↗
PubMed2001

Exploiting molecular mimicry to broaden the immune response to carbohydrate antigens for vaccine development.

Peptide mimetics of carbohydrates represent an alternative approach to induce anti-carbohydrate responses. Depending on their formulation, peptide mimetics can mediate T-independent or T-dependent responses. Multivalent peptide mimeotopes can induce high IgM/IgG ratios, as non-conjugated carbohydrates do. Here we observe that immunization with multivalent peptide mimeotope conjugated to BSA enhances carbohydrate reactive antibodies in Balb/c mice and xid mice, with IgG1 greater than IgG2a, in xid mice. These results suggest that mimeotope-conjugate formulations might augment carbohydrate-specific immune responses in immuno-compromised hosts.

Amino Acid Sequence↗
PubMed2001

Axonal projections and synapses from the supratrigeminal region to hypoglossal motoneurons in the rat.

Neural circuits from the supratrigeminal region (Vsup) to the hypoglossal motor nucleus were studied in rats using anterograde and retrograde neuroanatomical tracing methodologies. Iontophoretic injection of 10% biotinylated dextran amine (BDA) unilaterally into the Vsup anterogradely labeled axons and axon terminals bilaterally in the hypoglossal nucleus (XII) as well as other regions of the brainstem. In the ipsilateral XII, the highest density of BDA labeling was found in the dorsal compartment and the ventromedial subcompartment of the ventral compartment, where BDA labeling formed a dense, patchy distribution. Microinjection of 20% horseradish peroxidase (HRP) ipsilaterally or bilaterally into the tongue resulted in retrograde labeling of XII motoneurons confined to the dorsal and ventral compartments of the hypoglossal motor nucleus. Under light microscopical examination, BDA-labeled terminals were observed closely apposing the somata and primary dendrites of HRP-labeled hypoglossal motoneurons. Two hundred and sixty-five of these BDA-labeled terminals were examined at the ultrastructural level. One hundred and twelve BDA-labeled axon terminals were observed synapsing with either the somata (39%, 44/112) or the large or medium-size dendrites (61%, 68/112) of retrogradely labeled hypoglossal motoneurons. Axon terminals containing spherical vesicles (S-type) formed asymmetric synapses with HRP-labeled hypoglossal motoneuron dendrites. In contrast to this, F(F)-type axon terminals, containing flattened vesicles, formed symmetric synapses with both the somata and dendrites of HRP-labeled hypoglossal motoneurons with a preponderance of the contacts on their somata. Axon terminals containing pleomorphic vesicles (F(P)-type) were noted forming both symmetric and asymmetric synapses with HRP-labeled hypoglossal motoneuron somata and dendrites. The present study provides anatomical evidence of neuronal projections and synaptic connections from the supratrigeminal region to hypoglossal motoneurons. These data suggest that the supratrigeminal region, as one of the premotor neuronal pools of the hypoglossal nucleus, may coordinate and modulate the activity of tongue muscles during oral motor behaviors.

Animals↗
PubMed2001

Elucidation of neuronal circuitry: protocol(s) combining intracellular labeling, neuroanatomical tracing and immunocytochemical methodologies.

We describe a protocol combining either intracellular biotinamide staining or anterograde biotinylated dextran amine (BDA) tracing with retrograde horseradish peroxidase (HRP) labeling and immunocytochemistry in order to map physiologically identified neuronal pathways. Presynaptic neurons including their boutons are labeled by either intracellular injection of biotinamide or extracellular injection of BDA while postsynaptic neurons are labeled with HRP via retrograde transport. Tissues are first processed to detect HRP using a tetramethylbenzidine and sodium-tungstate method. Biotinamide or BDA staining is then visualized using an ABC-diaminobenzidine-Ni method and finally the tissue is immunocytochemically stained using choline acetyltransferase (ChAT) or parvalbumin antibodies and a peroxidase-anti-peroxidase method. After processing, biotinamide, BDA, HRP and immunocytochemical staining can readily be distinguished by differences in the size, color and texture of their reaction products. We have utilized this methodology to explore synaptic relationships between trigeminal primary afferent neurons and brainstem projection and motoneurons at both the light and electron microscopic levels. This multiple labeling methodology could be readily adapted to characterize the physiological, morphological and neurochemical properties of other neuronal pathways.

Animals↗
PubMed2001

Molecular cloning and functional identification of (+)-delta-cadinene-8-hydroxylase, a cytochrome P450 mono-oxygenase (CYP706B1) of cotton sesquiterpene biosynthesis.

In cotton, gossypol and related sesquiterpene aldehydes are present in the glands of aerial tissues and in epidermal cells of roots. A cytochrome P450 was found to be expressed in aerial tissues of glanded cotton cultivars, but not or at an extremely low level in the aerial tissues of a glandless cultivar. Its cDNA was then isolated from Gossypium arboreum L. After expression in Saccharomyces cerevisiae, the P450 was found to catalyse the hydroxylation of (+)-delta-cadinene, forming 8-hydroxy-(+)-delta-cadinene. This P450 mono-oxygenase has been classified as CYP706B1, and is the first member of the CYP706 family for which a function has been determined. Sesquiterpene aldehydes and CYP706B1 transcripts were detected in roots of both the glanded and glandless cultivars and in aerial tissues of the glanded cultivar. In suspension cultured cells of G. arboreum, elicitors prepared from the phytopathogenic fungus Verticillium dahliae caused a dramatic induction of CYP706B1 expression. The expression pattern of CYP706B1 and the position at which it hydroxylates (+)-delta-cadinene suggest that it catalyses an early step in gossypol biosynthesis. Southern blotting revealed a single copy of CYP706B1 in the genome of G. arboreum. CYP706B1 holds good potential for manipulation of gossypol levels in cottonseed via genetic engineering.

Chromatography, High Pressure Liquid↗
PubMed2001

Immunization with a carbohydrate mimicking peptide augments tumor-specific cellular responses.

The metastatic potential of some tumor cells is associated with the expression of the neolactoseries antigens sialyl-Lewis x (sLex) and sialyl-Lewis a (sLea) as they are ligands for selectins. We have recently shown that peptide mimetics of these antigens can potentiate IgG2a antibodies, which are associated with a Th1-type cellular response. As L-selectin is preferentially expressed on CD4+ Th1 and CD8+ T cell populations, specific induction of these phenotypes could augment a response to L-selectin ligand-expressing tumor cells. Here we demonstrate that immunization with a multiple antigen peptide (MAP) mimetic of sugar constituents of neolactoseries antigens induces a MHC-dependent peptide-specific cellular response that triggers IFN-gamma production upon peptide stimulation, correlating with IgG2a induction. Surprisingly, T lymphocytes from peptide-immunized animals were activated in vitro by sLex, also triggering IFN-gamma production in a MHC-dependent manner. Stimulation by peptide or carbohydrate resulted in loss of L-selectin on CD4+ T cells confirming a Th1 phenotype. We also observed an enhancement in cytotoxic T lymphocyte (CTL) activity in vitro against sLex-expressing Meth A cells using effector cells from Meth A-primed/peptide-boosted animals. CTL activity was inhibited by both anti-MHC class I and anti-L-selectin antibodies. These results further support a role for L-selectin in tumor rejection along with the engagement by the TCR for most likely processed tumor-associated glycopeptides, focusing on peptide mimetics as a means to induce carbohydrate reactive cellular responses.

Amino Acid Sequence↗
PubMed2001

Exploiting molecular mimicry: defining rules of the game.

Molecular mimicry has been touted as a mean to develop new generation of vaccines to target carbohydrate antigens on pathogens and on tumor cells. Structural and immunological rules governing molecular mimicry require definition for its successful exploitation. Of interest are the kinds of structures that peptides adopt as carbohydrate mimics, the extent to which topological or sequence similarities among peptide mimeotopes define serum cross-reactivity to carbohydrate antigens and the extent to which peptide mimeotopes affect T-cell responses. Rational design concepts can be applied to define how a peptide may mimic carbohydrate antigens, similarities in binding affinities of antibodies for carbohydrate and for peptides, how peptides can mimic core structures on otherwise dissimilar carbohydrate antigens, and how peptide mimeotopes can be used to manipulate cellular responses not achievable by carbohydrate antigens.

Amino Acid Sequence↗
PubMed2001

[The effect of heparin on the deep second-degree burn in scalded rats].

OBJECTIVE: To investigate the effects of heparin on deep second-degree burn in scalded rats. METHODS: Thirty Sprague-Dawley rats inflicted with 20% TBSA deep second-degree scalding on the back were employed as the model and were randomly divided into two groups. i,e heparin (H) and normal saline groups (N). Heparin (100 U . kg(-1) . day(-1)) was injected subcutaneously to the rats in H group while normal saline to those in N group once a day. Wound healing rate and immunological indices were determined simultaneously. RESULTS: (1) The wound healing time was much shorter in H group than that in N group (P < 0.005). The granulation tissue growth condition examined by LM and EM was much better in H group that than in N group. (2) The lymphocytic transforming ratio in H group was evidently increased (P < 0.005). And the phagocytic function of the macrophages increased significantly (P < 0.05). But there was no difference between the two groups in terms of hemoltytic plaque test (P > 0.05). CONCLUSION: Injection of heparin subcutaneously could accelerate burn wound healing and strengthen the systemic cellular immune potential and the macrophage phagocytic capacity of the host, but exerted no effect on the humoral immunity.

Animals↗
PubMed2000

Escape from gene silencing in ICF syndrome: evidence for advanced replication time as a major determinant.

Chromosomal abnormalities associated with hypomethylation of classical satellite regions are characteristic for the ICF immunodeficiency syndrome. We, as well as others, have found that these effects derive from mutations in the DNMT3B DNA methyltransferase gene. Here we examine further the molecular phenotype of ICF cells and report several examples of extensive hypomethylation that are associated with advanced replication time, nuclease hypersensitivity and a variable escape from silencing for genes on the inactive X and Y chromosomes. Our analysis suggests that all genes on the inactive X chromosome may be extremely hypomethylated at their 5' CpG islands. Our studies of G6PD in one ICF female and SYBL1 in another ICF female provide the first examples of abnormal escape from X chromosome inactivation in untransformed human fibroblasts. XIST RNA localization is normal in these cells, arguing against an independent silencing role for this RNA in somatic cells. SYBL1 silencing is also disrupted on the Y chromosome in ICF male cells. Increased chromatin sensitivity to nuclease was found at all hypomethylated promoters examined, including those of silenced genes. The persistence of inactivation in these latter cases appears to depend critically on delayed replication of DNA because escape from silencing was only seen when replication was advanced to an active X-like pattern.

Alleles↗