Institute of Biomedical Studies

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The recombinant expression of two pollen allergens using plant-viral and yeast expression systems.
(2006-05-09T19:12:08Z) Moehnke, Marcie H.; Kearney, Christopher Michel, 1958-; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Allergic disease causes much distress within industrialized areas of the world, affecting approximately a quarter of the population in such areas. Current immunotherapy involves the administration of increasing concentrations of crude allergen extracts over a period of time, in an attempt to switch the individual's allergic response to that of a non-allergic individual. Such therapy is largely ineffective, especially for cedar hypersensitivity where only 30% of individuals respond after two years of weekly injections, and unwanted and sometimes life-threatening side effects can accompany specific immunotherapy. In an effort to increase its efficacy, as well as circumvent these negative side effects, recombinant DNA technology is being used to produce recombinant allergens that will take the place of crude allergen extracts found in immunotherapy injections. A main cause of allergic disease within south central Texas is pollen produced by mountain cedars, Juniperus ashei. In one study, I cloned a particular mountain cedar allergen, Jun a 3, into a tobacco mosaic virus vector under the regulation of a subgenomic promoter. Infectious viral transcripts were inoculated onto Nicotiana benthamiana plants, and recombinant Jun a 3 protein was detected within these plants at 21 days post-inoculation. The recombinant protein was able to bind anti-Jun a 3 IgG antibodies as well as IgE from mountain cedar allergic patient sera. A separate study also demonstrated the successful expression of recombinant Jun a 3, but in a yeast expression system. The Jun a 3 cDNA was cloned into a yeast expression vector and transformed into Pichia pastoris cells for expression. Western blotting and ELISA experiments confirmed the recombinant Jun a 3 produced by the yeast bound anti-Jun a 3 IgG antibodies and IgE from allergic patient sera. A third study utilized the tobacco mosaic virus-based plant expression system to produce the main Italian cypress allergen, Cup s 1, from Cupressus sempervirens. This recombinant allergen behaved very similarly to a native cross-reactive allergen in its binding to IgG antibodies and allergic patient sera.
Gamma Hydroxybutyrate (GHB): mechanisms of central nervous system toxicity.
(2006-07-31T01:14:52Z) Lyng, Eric E.; Bottiglieri, Teodoro, 1958-; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Gamma Hydroxybutyrate (GHB) is an endogenous metabolite of gamma-aminobutyric acid (GABA) and a putative neurotransmitter found in mammalian brain. The illicit use of GHB is a growing health care concern in the U.S. Low doses have euphoric and stimulatory effects while high doses act as a CNS depressant and can cause respiratory failure. In addition to fatalities and drug facilitated rape, in 2004 over 7000 GHB overdoses were reported in the U.S. Gamma Butyrolactone (GBL) and 1,4-Butanediol (1,4-BD), precursors of GHB, can cause similar effects after being converted to GHB in the body. While GHB is a Schedule I compound, recently it was given a Schedule III classification as a drug for treatment of cataplexy associated with narcolepsy. Individuals affected by the inherited disorder succinic semialdehyde dehydrogenase (SSADH) deficiency have significant elevation of GABA and GHB in body tissues, and a range of neurological complications. Currently there is no treatment option for a GHB overdose situation or for SSADH patients. GHB has been shown to inhibit striatal dopamine release leading to sedation and loss of locomotor activity in rodents. However, GHB’s mechanism of action is poorly understood. This dissertation investigated acute and chronic effects of GBL, a precursor to GHB, on locomotor function and monoamine neurotransmitter metabolism in mice. Dose response studies were performed to characterize the effects of GBL. Compounds aimed at increasing central dopaminergic activity or antagonism of GABAergic activity were tested for their ability to antagonize the locomotor effects induced by GBL. In total, eight different compounds were studied of which pergolide and nomifensine were successful at antagonizing the loss of locomotor activity when administered either prior to or after GBL. Chronic administration of GBL over the course of 14 days was evaluated in mice using a rotarod system. These studies did not reveal any long term detrimental effect of GBL on locomotor function. This dissertation is the first report showing the ability of pergolide and nomifensine to antagonize the loss of locomotor activity induced by GBL. These studies provide insight into treatment options for GHB toxicity or overdose as well as for patients with SSADH deficiency.
Anti-tumor activity of an oncolytic adenoviral construct expressing a small interfering RNA transgene.
(Philadelphia, PA : American Association for Cancer Research., 2006-10-01) Samuel, Shirley Kulangara.; Tong, Alex W.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Cancer is a leading cause of mortality in the world today. Mutation in the K-ras oncogene is common in most human cancers. K-ras oncogene expression was specifically downregulated by 58.7% by K-ras silencing siRNA, and this was accompanied by 66% growth inhibition of NCI-H441 lung cancer cells. To improve siRNA delivery and cause its stable expression in cancer cells, we used ONYX-411, an oncolytic adenovirus as a backbone to clone the K-ras silencing siRNA. This new adenoviral construct called Internavec, significantly downregulated mutant K-ras expression by 48.9% (p<0.05, n=3) as compared with 11.9% downregulation by parental ONYX-411 in HTB-79 pancreatic cancer cells. The anti-tumor activity of Internavec was examined in various cancer cell lines with and without the relevant K-ras mutation to observe the specificity of the siRNA transgene against the glycine to valine mutation on codon 12. Internavec showed enhanced anti-tumor activity in cell lines with the relevant mutation, compared with ONYX-411. Internavec (5 @ 1x108 pfu) significantly reduced the growth of subcutaneous HTB-79 pancreatic tumor xenografts in vivo by 85.5%, including complete growth suppression in 3 of 5 mice. Parental ONYX-411 or ONYX-411-siRNAGFP was markedly less effective (47.8% and 44.1% growth reduction, p<0.05, respectively). To characterize interferon-inducing activity of Internavec, landmark gene expression of the interferon pathway (OAS1, MX1) was examined following Internavec treatment, using HEK-293 cells as positive control. HEK-293 cells displayed an upregulation of OAS1 and MX1 following Poly (I:C) treatment. However, Internavec did not upregulate these interferon-pathway genes in HEK-293 or H79 lines, suggesting a lack of interferon activation by Internavec. To delineate underlying molecular events contributing to the enhanced growth inhibition, microarray experiments were performed on cells treated with Internavec. Internavec, but not ONYX-411, downregulated the expression of multiple Ras signaling-related genes (MAP4K5, PLCε1, IKBKB, FOXO3A and RAB28). These findings indicate that the knockdown of mutant K-ras serves to enhance oncolytic virus anti-tumor activity through the perturbation of additional cellular signaling events.
Growth inhibition of human multiple myeloma cells by a conditional-replicative, oncolytic adenovirus armed with the CD154 (CD40-ligand) transgene.
(2007-03-08T15:37:14Z) Rodrigues, Margret S.; Tong, Alex W.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Human multiple myeloma (MM) remains incurable despite successes in induction therapy. Based on our previous findings that treatment with the recombinant CD40L protein inhibited the growth of CD40+ human myeloma cell lines, we examined the targeting of the myeloma cell surface receptor, CD40 by its natural ligand, CD40L, using a gene transfer approach. For targeted delivery of CD40L, we have constructed a conditional-replicative adenoviral for delivery of the CD40L transgene (AdEHCD40L). AdEHCD40L incorporates tumor/tissue specific promoters limiting transgene expression and viral replication to HIF (hypoxia-inducing factor)-1α and/or estrogen overexpressing cancer cells. Conditional expression of the early adenoviral E1A gene and the CD40L transgene was validated in the IL-6 independent MM line RPMI 8226 (62% and 66%, respectively), and the IL-6 dependent cell line Kas-6/1 (32.7% and 30%, respectively). Treatment with AdEHCD40L resulted in pronounced growth inhibition (RPMI 8226: 95.5 ± 2.1%; Kas-6/1: 80.5 ± 9.8%, mean ± SD), and was more effective than the parental construct without the CD40L transgene (AdEHNULL)(p=0.04). Both AdEHNULL and AdEHCD40L were minimally cytotoxic to normal peripheral blood mononuclear cells and IMR-90, normal fibroblast cells (2.8 ± 0.3%). We also observed a 53% reduction in myeloma xenograft growth, following AdEHCD40L intratumoral injections (4.8 ± 0.9 mm, vs. 10.5 ± 1.2 mm in mock-treated animals; p=0.002), which was more effective than AdEHNULL (7.6 ± 1.1 mm; p=0.03). Evidence of adenoviral hexon and CD40L transgene expression within the treated tumors was demonstrated. In the study of cellular events leading to myeloma growth inhibition, we observed increased apoptotic activity at 72 hrs following treatment with AdEHCD40L infection (21.3 ± 6.5% vs. 7.7 ± 1.9% in untreated; p=0.007) which was accompanied by increased S phase accumulation (68.0 ± 2.4%, vs. 54.0 ± 5.5% in AdEHNULL and 48.9 ± 4.6% in untreated culture; p<0.003). High throughput gene array expression analysis identified pro-apoptotic TNF superfamily members, Fas and TRAIL and cytokines, IL-8 and RANTES to be uniquely upregulated by AdEHCD40L. These findings confirm the growth inhibitory activity of AdEHCD40L, through the induction of apoptosis and is a promising approach for the experimental gene therapy of human multiple myeloma.
Design, optimization, and evaluation of conditionally active gene therapy vectors.
(2008-06-09T11:51:54Z) Wood, David Rowe.; Ding, Jiahuan.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
It is the objective of this project to produce gene therapy vectors that are active and/or significantly up-regulated due to specific physiological conditions. The significance of such constructs is that it imparts a greater degree of control in the implementation of gene therapy. In general, it is desirable for a gene therapy vector to be active only when and where it is needed. The majority of gene therapy research to date has focused primarily on obtaining expression levels high enough to elicit a therapeutic response, as well as, distributing the vector to enough tissues to provide a corrective effect to the disorder being addressed. However, simply having a gene adequately delivered to enough cells to treat disease and having the gene product be produced in sufficient amounts to have a therapeutic effect cannot be the end of the story. Not unlike genes found naturally in the body, artificially delivered genes also need to be regulated. The construction of such vectors could prove useful for the treatment of disorders; such as Coronary Artery Disease (CAD) or even Cardiomyopathy, that occur in a specific tissue type or that are associated with an abnormal physiological state, such as hypoxia. Our vector constructs are a small step towards this ultimate goal. In this study, we present data on DNA vectors that were designed, constructed and evaluated in vitro and in vivo; both qualitatively and quantitatively. We report success in the creation of vectors/plasmids that are primarily cardiac tissue specific (pMHCI, pMHCII), vectors that are regulated by cellular oxygen levels (pHAL, pHAM), and even some success in combining the two (pHMHC).
Humanized mice to test vaccination against influenza virus via dendritic cells.
(2008-06-11T14:19:03Z) Yu, Chun-I, 1975-; Palucka, Karolina.; Banchereau, Jacques.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Critical to the development of human vaccines is the availability of in vivo models of the human immune system that permit testing of vaccine efficacy. Here,we used NOD-SCID-β²m-/- immunodeficient mice which, when engrafted with human CD34⁺ hematopoietic progenitors, develop all subsets of human dendritic cells(DCs) and B cells. T cells and their subsets were reconstituted by adoptive transfer. We found myeloid DCs, plasmacytoid DCs and monocytes in the bone marrow, spleen, and peripheral tissues including skin and lungs. To test DC biologyin vivo, we first used live influenza A/PR8/34 (H1N1) virus. Upon intranasal inoculation, all subsets of human antigen presenting cells were activated. Matured DCs were found accumulated in mediastinal lymph nodes. To evaluate the value of these mice for testing human vaccines, humanized mice were immunized with1) ex vivo-generated DCs, 2) seasonal influenza vaccines and 3) protein antigens fused to anti-DC receptor. Upon vaccination with ex vivo-generated DCs pulsed with heat-inactivated influenza virus, mice developed influenza-specific immunity, i.e. influenza-specific immunoglobulins (Igs) in the serum and influenza virus matrix protein 1 (FluM1)-specific CD8⁺ T cells in the blood, spleen and lungs. Influenza-specific Igs were protective as sera from vaccinated mice inhibited influenza virus-induced hemagglutination in vitro and offered passive protection in vivo. Upon vaccination with seasonal influenza vaccines, i.e. live attenuated trivalent vaccine (LAIV) or killed trivalent vaccine (TIV), humanized mice developed both humoral and cellular immunity. Plasma cells differentiation and the secretion of specific Igs were dependent on the reconstitution with CD45RA⁻CD27⁺CD4⁺ central memory T cells. CD8⁺ T cells specific to two influenza antigens, i.e. FluM1 and NS1, were detected in mice vaccinated with LAIV. TIV-vaccinated mice showed the expansion of FluM1, but not NS1, specific CD8⁺ T cells. Antigen-specific CD8⁺ T cells produced IFN-γ and expressed surface CD107a consistent with the acquisition of effector function. Finally, upon vaccination with anti-DC receptor (DCIR)-FluM1 fusion protein and poly I:C as an adjuvant, DCs efficiently cross-presented FluM1 and expanded antigen-specific CD8⁺T cells. Therefore, humanized mice might be valuable model for testing human vaccines against influenza virus.
Gene expression profiling to understand the alterations in the monocyte compartment of pediatric systemic lupus erythematosus.
(2008-06-11T17:41:33Z) Patel, Pinakeen Shankarbhai.; Pascual, Virginia.; Banchereau, Jacques.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Blood monocytes from SLE patients display DC function, as they are able to induce the proliferation of allogeneic T cells. Furthermore, sera from SLE patients induce healthy monocytes to differentiate into DCs. This DC-inducing property is in part due to the presence of type-I IFNs in SLE sera, as well as other, yet uncharacterized factors. To understand these alterations, we performed a thorough phenotypic analysis and gene expression profiling of monocytes from children with active, newly diagnosed and untreated disease. Phenotypic analysis of freshly isolated SLE blood monocytes revealed a modest expansion of CD14highCD16+ cells and an otherwise lack of expression of molecules related to DC function. Further characterization of a fraction of SLE monocytes inducing allogeneic T cell proliferation revealed that upon contact with T cells, SLE monocytes secrete proinflammatory cytokines such as IL-1 and IL-6 and do upregulate expression of innate immunity receptors involved in DC differentiation and molecules responsible for antigen presentation. To recapitulate the initial events leading to monocyte differentiation in this disease, we studied the effects of SLE serum on healthy monocyte at the trasncriptional and protein levels. These studies revealed the upregulation of expression on these cells of chemokine receptor such as CX3CR1 and CCR7, which may lead to the migration of blood monocytes to inflammed tissues and/or secondary lymphoid organs respectively in vivo. There, contact with T cells would lead to the acquisition of antigen presenting function and skewing from tolerogenic to immunogenic responses.
Automated sequence homology : using empirical correlations to create graph-based networks for the elucidation of protein relationships.
(2008-10-02T18:47:42Z) Bush, Stephen J.; Baker, Erich J.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Identification of sequence homology has presented a formidable obstacle despite significant increases in both technological capability and detailed knowledge of genomes and proteomes. While PSI-BLAST remains the popular tool for the job, it often returns inaccurate results with unacceptable levels of false positives. In order to increase the sensitivity and accuracy of homology finding, we have developed a software application called Automated Sequence Homology that bypasses these shortcomings and provides reliable and precise results. The system presented here is based upon the creation of a graph-based network highlighting the relational connections between proteins using empirical correlations. It takes a step back from PSI-BLAST to the acclaimed BLAST algorithm to create a sampling of the protein relational network.
Analysis of inflammatory changes in human pancreatic islet cells.
(2009-02-27) Jackson, Andrew M.; Naziruddin, Bashoo.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Pancreatic islet cell transplantation is a promising investigational research treatment for labile type 1 diabetes mellitus. However, several obstacles still exist to the implementation of clinical islet cell transplantation as a standard therapy. These obstacles include a shortage of donor pancreata, an imperfect islet isolation procedure, significant loss of islets during the peri-transplant period, and islet toxicity of immunosuppressive drugs. The present study is focused on addressing two of these major hurdles; namely, identification of factors affecting the islet isolation, and understanding the mechanism involved in the peri-transplant loss of islets. Initial analysis was centered on the effect of cold ischemia time (CIT) on the islet isolation outcome. Comparison of varying CIT and several factors that determine the quantity and quality of islets obtained from 52 isolations was performed. This analysis showed that CIT of less than four hours significantly improved islet isolation results. This finding could help improve the current strategy used in clinical islet transplantation. We hypothesized that following transplantation, the inflammatory environment will specifically alter gene expression in transplanted islets, and also will induce surface expression of HLA-class II molecules. This, in turn, could cause anti-donor response resulting in islet destruction. To test the above hypotheses, two studies were performed. Islet cells were treated with control or type 1 diabetic serum. Gene expression was then analyzed using micro array and confirmed by real-time PCR. Islets treated with diabetic serum demonstrated specific induction of multiple genes reported to have secondary roles in angiogenesis while inhibiting transcription of genes with protective attributes against environmental stresses. Islet cells were treated with IFNγ and TNFα and analyzed for HLA class II induction by real-time PCR analysis, flow cytometry, and immunofluorescent imaging.Cytokine treated islets demonstrated significantly upregulated HLA class II gene transcription and surface expression. Importantly, islet transplant recipient serum showed increased binding and cytotoxicity specifically directed against cytokine treated islets. Together these data suggest that in the context of hepatic portal vein transplantation, islet cells contribute to the innate and adaptive immune response during the peri-transplant period resulting in islet destruction.
To giveth and taketh away : determination of taurine's protective role during ethanol withdrawal through supplementation and depletion paradigms.
(2009-04-01T19:40:25Z) Zalud, André W.; Diaz-Granados, Jaime L.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
The β-amino acid taurine, whose primary physiological roles involve osmoregulation and calcium modulation, exists as one of the most concentrated amino acids in the mammalian brain. As a neuroinhibitor and neuromodulator, taurine may provide a unique therapeutic approach in reducing the aversive symptoms of ethanol withdrawal that often promote further ethanol abuse. Taurine’s properties could efficiently defend sensitive neural tissues from disturbances of osmolarity and excitability, which typically manifest during episodes of ethanol withdrawal. This investigation examined taurine’s influence on ethanol withdrawal by testing the effects of both taurine treatment and taurine depletion on withdrawal-related convulsions, as well as associated neurochemical alterations in brain tissue. Results showed that taurine, administered during ethanol withdrawal, significantly reduced overall withdrawal severity in adult male C3H/HeJ mice. Conversely, significantly reduced tissue taurine levels, achieved via the taurine uptake inhibitor guanidinoethane sulfonate (GES), exacerbated the severity of ethanol withdrawal. Analysis of extracted tissues (pituitary, supraoptic nucleus of the hypothalamus, and hippocampus) suggests that taurine’s capacity to reduce withdrawal-related convulsions may result from an attenuated release of arginine vasopressin from both the pituitary and supraoptic nucleus. During ethanol withdrawal, conditions of over-hydration can lead to lower hippocampal osmolarities, which itself can produce hyperexcitable states. By inhibiting over-activated vasopressin outflow during withdrawal, taurine seemingly prevents systemic osmotic disruptions that would otherwise disturb osmolarities within sensitive hippocampal tissues. During ethanol withdrawal, treatment with exogenous taurine provides a significant reduction in withdrawal severity and may facilitate the recovery from alcoholism. Given that the current findings also reveal that taurine depletion severely aggravates the severity of ethanol withdrawal, both behaviorally and biochemically, alcohol recovery programs should consider the potential therapeutic benefits of taurine supplementation in treatment regimens.
Identification of dysregulated miRNA targets in human prostate cancer.
(2010-02-02T19:49:42Z) Chen, Po-hsun.; Tong, Alex W.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
MicroRNAs (miRNAs) are small, non-coding RNAs (18~24 nt) that regulate diverse cell functions in mammalian cells, including proliferation, differentiation, metabolism, stress resistance, and death. There is increasing evidence that altered expression and function of regulatory miRNAs contribute to the uncontrolled growth of human cancers. We examined the hypothesis that miRNA expression profiles can distinguish malignant from normal tissues. With the use of high throughput, liquid phase hybridization analyses (mirMASA) were carried out with 40 Stage 1c/2 prostate cancer specimens, using 114 miRNA-specific probes. We identified 5 miRNAs (miR -23b, -100, -145, -221, and -222) that were significantly downregulated in malignant prostate tissues as compared with their normal counterpart from the same patient. Decreased expression was confirmed by quantitative real-time PCR (qRT-PCR) analyses. The pathophysiological role of these downregulated miRNAs was further characterized in the prostate cancer LNCaP cell line, which exhibited similarly reduced expression of miR- 23b, -145, -221, and -222. Ectopic expression of mature miR-23b and miR-145 reduced LNCaP cell growth by >40%. In contrast, transfection with a miRNA (miR-141) that was upregulated did not markedly affect cancer cell growth. The altered expression of miR- 145 was of particular interest, in view of its similarly reduced expression in breast, cervical, colon, and lung carcinomas. In silico search (TargetScan) yielded 396 eligible targets for miR-145, of which 6 were markedly upregulated in LNCaP cells. These include the prostaglandin F receptor (PTGFR), transforming growth factor (TGFBR2), p21-activated kinase 7 (PAK7), SLIT-ROBO Rho GTPase activating protein 2 (SRGAP- 2), kinesin family member 3A (KIF3A), and Ras association (RalGDS/AF-6) domain family 2 (RASSF2). miR-145 was uniformly downregulated in all prostate caner lines tested (LNCaP, 22Rv1, PC-3, Du-145, as compared with the nonmalignant prostate line RWPE-1) This miRNA may be differentially expressed in androgen dependent (AD) and androgen independent (AI) prostate cancer cell lines according to qPCR analysis. Mean expression level of miR-145 was approximately 6-fold lower in AD (LNCaP, 22Rv1) than AI (PC3, Du-145) lines. qPCR analysis suggests that PTGFR and RASSF2 were upregulated in 3 of 4 prostate cancer cell lines, whereas MYCN was only upregulated in androgen independent cell lines. The expression of CCND2, MAP3K3 and MAP4K4 were not significantly upregulated by qPCR analysis. These findings suggest that altered miR-145 expression may impact prostate cancer growth through its loss of regulation of PTGFR and/or RASSF2 activities. qPCR analysis of miR-145 treated prostate cancer cells showed decreased expression of PTGFR and RASSF2 indicating these two genes are potential targets of miR-145 modulation. Direct binding only showed moderate reduction of RASSF2 and a more pronounced reduction of PTGFR. These studies suggest that miR-145 may modulate prostate cancer cell growth by modulating PTGFR activity.
RNAi screen for novel components in Caenorhabditis elegans ovulation and fertility.
(2010-02-02T19:59:00Z) Miles, Jonathan P.; Lee, Myeongwoo.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
The intercellular and intracellular signaling pathways elucidated through research on the nematode C. elegans provide valuable information on the communication systems throughout all organisms. Through the use of RNA interference (RNAi), it is possible to discover additional genes that may play roles in signaling pathways. The inositol trisphosphate (IP₃) signaling pathway maintains the basal and ovulatory contractions of the sheath cells in all C. elegans organisms. Utilizing an RNAi feeding protocol to knock down expression of genes, some 155 genes capable of causing sterility in wild-type C. elegans were identified. Focusing on these contractions of the sheath cells through control of the IP₃ signaling pathway, a mutant C. elegans for the IP₃ receptor, itr-1(sy290), was used. The ITR-1 receptor, located on the endoplasmic reticulum, normally allows for the release of calcium ions when IP₃ binds, and is constitutively active in the itr-1(sy290) mutant worm. The mutant itr-1(sy290) worms maintain higher concentrations of cytoplasmic calcium, which resulted in a rescue of the sterility seen in the wild-type worms in this study. Due to the potential for pleiotropic effects of many of these sterility causing genes, we looked for known components of the IP₃ signaling pathway (eg. plc-3) and at their sterility scores, as well as the scores most comparable to these known components. This reduced the gene pool down to 24 significant genes. Examination of these genes reveals a wider communication network necessary for proper ovulation in C. elegans.
Respiratory Syncytial Virus triggers immune tolerance through induction of tolerogenic dendritic cells and expansion of regulatory T cells.
(2010-06-23T12:28:05Z) Guo, Haisu.; Connolly, John Edward.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Dendritic cells have the ability to control the balance between immunity and tolerance. Upon viral exposure, Dendritic Cells (DCs) steadily detect pathogens and exert their antigen presentation function to induce adaptive T cell response. Respiratory Syncytial Virus (RSV) is an important respiratory pathogen in infants and young children worldwide. Here we show that RSV exposure polarizes DC maturation to a tolerogenic state. RSV exposed DCs (RSV-DCs) are unable to prime allogeneic CD4+ T cell proliferation and cytokine production. Strikingly, RSV exposed DCs are able to efficiently inhibit on-going Mixed Leukocyte Reaction (MLR) in trans. Phenotypic characterization of RSV-DCs indicates that they express a variety of surface inhibitory molecules and secrete high amount of the cytokine IL-10. Autocrine IL-10 receptor signaling is required for tolerogenic conversion. A direct comparison with pharmacologically generated tolerogenic DCs indicates RSV-DCs are much more potent at inhibiting CD4+ T cell alloproliferation. Furthermore, we find that RSV-DCs propagate their tolerogenic signal through expansion of regulatory T cells (Tregs). RSV- DCs induce the selective expansion of CD4+/CD25+/FoxP3+/CTLA+/GITR+ Tregs in the bulk T cell population. These Tregs are able to inhibit on-going MLR in trans, indicating their functional potency. An analysis of the non-proliferating target CD4+ T cells indicates that they are in a state of phenotypic and functional anergy. These T cells express anergy markers and are unresponsive to secondary anti-CD3/CD28 restimulation. Interaction of B7 negative co-stimualtor PD-L1 and its receptor PD-1 are required for Treg expansion and function as blockade led to a reversal of anergy induction in the target population. These in vitro observations led us to investigate the impact of RSV-DCs on immune tolerance in vivo. We did preliminary studies demonstrating RSV exposure induces BALB/c bone marrow derived DCs (BM-DCs) tolerogenic characterized by inhibition of C57BL/6 CD4 T cell alloproliferation and upregulation of mouse PD-L1 molecule. Similarly to what we observed in human cells, RSV-mouse DCs are able to expand a population of CD4+CD25+FoxP3+ regulatory T cells. These cells with immune suppressive function can then be adoptively transferred into murine models of autoimmune diseases and organ transplantion to suppress antigen-specific immune activation in vivo.
Molecular mechanisms in low-serotonin induced mood and anxiety disorders.
(2010-10-08T16:29:45Z) Tran, Lee.; Keele, N. Bradley.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
The amygdala has been shown to be involved in both epilepsy and emotional disturbances such as in mood and anxiety disorders. It is suggested that anxiety and mood disorders may be the result of sub-seizure hyperexcitability in limbic areas such as the amygdala. Epilepsy-like mechanisms involve increased glutamatergic activity, whereas low serotonin (5-hydroxytryptamine, 5-HT) is associated with abnormal emotion. Although much evidence suggests low 5-HT increases excitability, the molecular mechanisms underlying this process are not known. Here we explored the ability of low serotonin to increase glutamate receptor (GluR) expression resulting in increased anxiety-like behavior. Using qRT-PCR, we found that individually-housed rats treated with p-chlorophenylalanine (300 mg/kg i.p.), an inhibitor of tyrosine hydroxylase, resulted in 21.8 fold higher GluR1 mRNA expression in amygdala neurons. Similar results were found in rats treated with bilateral infusions of 5’7-dihydroxytryptamine (DHT, 8 μg/side) into the lateral nucleus of the amygdala (LA) resulting in a 10³ fold increase in GluR1 mRNA. Further, Western blot analysis confirmed an overall 50.0% increase in GluR1 protein expression without any significant increase in other GluR subunits. These results suggested that low serotonin induces hyperexcitability of LA neurons by increasing GluR1 mRNA, and led to increased expression of GluR1. Additionally, we showed that these molecular changes resulted in behavioral differences in the open field maze, but not the plus maze. Rats treated with 5,7-DHT had a higher degree of center avoidance. The signaling pathway involved was also of interest. We found that the increase in GluRs may be mediated by CaMKII as shown by a ~60% increase in CaMKII phosphorylation. In order to further investigate, RNAi delivered by an AAV vector was used to knock down CaMKII expression and the effects on GluR expression was assessed. We showed that knockdown of CaMKII resulted in up to 37% decrease in GluR1 mRNA. CaMKII knockdown was also able to reverse the anxiogenic effects of decreased 5-HT, increasing center entries up to 30% and center duration up to 40% compared to controls. These results suggested that low serotonin induced hyperexcitability in LA neurons by increasing GluR1 possibly through a CaMKII mediated pathway, and led to increased open-field anxiety. This mechanism could be important in the pathophysiology of mood and anxiety disorders.
The effect of dietary folate deficiency and age on methylation metabolites, neurotransmitters, and behavior in mice.
(2011-01-05T19:46:26Z) Wasek, Brandi Lynn.; Bottiglieri, Teodoro, 1958-; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Severe folate deficiency caused by inborn errors of metabolism has profound deleterious effects in the CNS such as mental retardation, psychiatric disorders, seizures and myelopathy. Mild folate deficiency, due to dietary insufficiency, drugs, or a common mutation in the gene encoding methylenetetrahydrofolate reductase (MTHFR C677T), are associated with an increased risk for depression and dementia, especially in the aging population. The metabolism of folate is intimately linked with the synthesis of S-adenosylmethionine (SAM), the sole source of methyl groups required in methyltransferase reactions. S-adenosylhomocysteine (SAH), a product of methylation reactions, accumulates in folate deficiency due to increased conversion from homocysteine. Recent studies have linked hyperhomocysteinemia and hypomethylation to gene activity, as well as methylation dependent post-translational modification of proteins and neurotransmitter metabolism in depression and dementia. To better define the role of folate deficiency in CNS function, we fed young and old C57BL/6J mice folate deficient diets for 3 months and old heterozygous tg-MTHFR mice a low folate diet for 6 months. Mice were tested for grip strength, coordination, open field activity, and spatial memory. After treatment mice were sacrificed by CO2 asphyxiation or microwave radiation. Blood, peripheral and regional brain tissues were processed for the analysis of methylation and neurotransmitter metabolites. Age did not influence brain methylation metabolites in C57BL/6J mice. Low folate and folate deficient diets decreased the SAM/SAH ratio, an indicator of methylation status in most brain regions from C57BL/6J and heterozygous tg-MTHFR mice. Overall, the effects of folate deficiency were not exacerbated by age in C57BL/6J mice. Brain methylation metabolites differed significantly depending on the method of sacrifice. Specifically, folate deficiency decreased SAM in mice sacrificed by microwave radiation and increased SAH in mice sacrificed by asphyxiation. Folate deficiency reduced dopamine and serotonin turnover in several brain regions although the levels of the parent neurotransmitters were unaffected. Choline and acetylcholine levels were reduced by folate deficiency in the mid-brain. Folate deficiency impaired open field behavior, but did not have any significant effect on spatial memory. These studies help to further our understanding of the mechanisms involved in folate deficiency on CNS function in the aging brain.
Development of dendritic cell-based vaccines for the treatment of HIV-infected patients.
(2011-09-14) Cobb, Amanda J., 1979-; Palucka, Karolina.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Dendritic cells (DCs) are professional antigen-presenting cells of the immune system with the ability to induce and control T and B cells responses. Monocytes have been used as precursors to generate DC vaccines ex vivo. Monocyte-derived DCs have been used as vaccines in clinical trials to treat cancer and infectious disease by eliciting potent T cell responses. Infection with Human Immunodeficiency Virus (HIV) almost invariably leads to a chronic disease. Eventually the destruction of the immune system culminates into Acquired Immune Deficiency Syndrome (AIDS). Since the advent of Highly Active Antiretroviral Therapy (HAART), HIV patients have been able to control viral levels. However, with prolonged use of treatment, these patients experience serious adverse effects such as liver and mitochondrial toxicity that can potentially become fatal. Long-Term Non-Progressors (LTNP) are a unique subpopulation of HIV patients that are able to control their viral load without HAART. Studies revealed HIV-specific polyfunctional CD8⁺ T cells to be vital for viral control. Thus, we have focused our efforts on devising therapeutic HIV DC vaccines to increase the quality of CD8⁺ T cell responses in chronic HIV infected patients. First, we conducted a series of preclinical development assays of GM-CSF/IFN-α and GM-CSF/IL-15 HIV DC vaccines loaded with a mixture of HIV-1-antigen lipopeptides (ANRS HIV-LIPO-5 vaccine), which is comprised of five HIV-1-antigen peptides (Gag₁₇₋₃₅, Gag₂₅₃₋₂₈₄, Nef₆₆₋₉₇, Nef₁₁₆₋₁₄₅, and Pol₃₂₅₋₃₅₅) each covalently linked to a palmitoyl-lysylamide moiety, and activated with LPS. Functional assays demonstrated that the DC vaccines expressed DC differentiation markers and displayed typical morphology which included the presence of dendrites important for cellular interaction. Potency assays proved the DC vaccines were capable of eliciting HIV-1-antigen-specific CD4⁺ and CD8⁺ T cell responses. Our studies have demonstrated the use of DC vaccines in eliciting HIV-specific CD8⁺ T cells responses in vitro. Furthermore, the pre-clinical development of the GM-CSF/IFN-α DC vaccine activated with LPS led to the evaluation of safety and immunogenicity in a Phase I/II clinical trial in chronically HIV-1-infected patients on HAART (clinicaltrials.gov identifier: NCT00796770).
Inhibition of β-lactamase I from Bacillus cereus by ssDNA.
(2011-09-14) Foster, Taylor Ann.; Kim, Sung-Kun, 1968-; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Inhibitors of β-lactamases are important to the treatment of infectious diseases when used in conjunction with a β-lactam antibiotic. Current inhibitors of β-lactamase such as clavulanic acid, sulbactam, and tazobactam perform efficiently overall but due to developing bacterial resistances to these inhibitors, new inhibitors need to be discovered. SELEX procedures were used to isolate ssDNA aptamers capable of binding to the enzyme active site and consequently inhibit the action of β-lactamase I from Bacillus cereus 569/H/9. A 22 base ssDNA aptamer was discovered to have an inhibition pattern consistent with reversible competitive inhibition. These results prompted further study of a hairpin loop of 10 bases and a linear 11 base ssDNA aptamer which were truncated forms of the original 22 base aptamer. The 11 base aptamer failed to show any inhibition against β-lactamase I, whereas the 10 base aptamer showed competitive reversible inhibition.
Leveraging available data for contaminants of emerging concern to develop an understanding of environmental hazard.
(2011-09-14) Berninger, Jason P.; Brooks, Bryan W.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Contaminants of emerging concern (CEC) are classes of compounds with relatively limited information available on environmental exposure, fate, and effects. The purpose of this research was to develop and test approaches that leverage available data using probabilistic models to advance an understanding of aquatic hazards of CECs. Pharmaceuticals are one such group of CECs. Though extrapolation approaches with fish models can provide important bridges between the biomedical and environmental sciences, little data is available addressing the sublethal effects of therapeutics in aquatic organisms. Seldom is the drug’s Mode of Action (MOA) considered in selection of chronic endpoints for an assessment, though mammalian pharmacological information is available for most drugs. A statistically significant relationship (r²=0.846, p<0.001) between mammalian pharmacology and toxicology data (margin of safety) and available fish acute to chronic data was identified, when therapeutic MOA was considered in selecting a chronic response variable. Based on this relationship, metrics to assess potency and internal effective dose were developed. These metrics were then evaluated using probabilistic distributions in an effort to prioritize drugs based on potential hazard. These probabilistic assessments identified specific drugs and drug classes as potentially presenting greater hazard to fish. To test these models, toxicity experiments with diphenhydramine, an antihistamine drug, were conducted to characterize standardized endpoints and novel, MOA-related ecotoxicological endpoints. The results confirmed that sublethal endpoints (e.g., behavior) related to therapeutic may be more appropriate for fish and that leveraging mammalian pharmacology and toxicology data may be predictive for MOA related responses when evolutionary conservation of targets are considered. It further highlighted the importance of carefully selecting model organisms for study of pharmaceuticals with multiple MOAs, because reproduction of the invertebrate Daphnia magna was sensitive to diphenydramine, potentially resulting from its histaminergic and cholinergic activities. A similar probabilistic approach was applied to oil dispersants, another CEC class, to assess potential impacts to aquatic systems. Leveraging the limited acute toxicity data available for an invertebrate and a fish model, probabilistic distributions were employed to predict the likelihood of oil dispersants exerting acute toxicity in the presence or absence of oil. This approach can be utilized in prospective and retrospective assessments to support emergency response decisions to oil spills and prioritize substances for further study. Lastly, probabilistic methods were used to develop uncertainty factors for acute to chronic rations for select biological active chemicals. For many chemical classes chronic effects data is lacking. Typically, default uncertainty factors are utilized to bridge this data gap. By leveraging the available chronic data using probabilistic methods, novel data-driven uncertainty factors were developed, potentially providing more protective extrapolation models.
Folding, dynamics and interaction studies of the Neuropeptide Y family.
(2011-12-19) Hegefeld, Wendy A.; Jas, Gouri Sankar.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
Detailed molecular knowledge of the complex dynamics of biological processes such as folding and the interaction with cellular membranes may greatly advance the treatment of human disease. The Neuropeptide Y family of peptides are highly conserved peptides found in nature, consist of 36 amino acids, and are widely distributed in the central nervous system of mammals. A detailed study of thermodynamics, kinetics, membrane translocation, and receptor interaction of human neuropeptide Y (NPY) and the thermodynamics and detailed molecular modeling of human peptide YY (PYY) were investigated. These studies employed a combination of experiment and simulation to characterize the two most important members of the neuropeptide Y family. The formation of secondary structures is one of the most fundamental processes in protein folding. To obtain a detailed understanding of protein folding it is useful to study peptide models that provide well defined stable structures in solution. NPY and PYY are composed primarily of two important secondary structural elements: the α-helix and hairpin-like structure. Therefore, further analysis of the three most prevalent helical secondary structures found in nature (α-, 3₁₀-, π-helix) and the β-hairpin structure were carried out with carefully designed peptide models to characterize their individual structures and formation. These studies suggest two different mechanisms of formation for helical and β-hairpin structures.
Structure, dynamics, and interactions of Angiotensin family of peptides with functional implications.
(2011-12-19) DeLeon, Kristine Y.; Jas, Gouri Sankar.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.
We present a study of structural analysis and reorientational dynamics of the angiotensin family of peptides. AngI is a decapeptide that acts as a precursor to the octapeptide, AngII in the Renin Angiotensin Aldosterone system for blood pressure regulation. Smaller metabolites of AngII, including AngIII, AngIV and Ang₁₋₇, have recently been discovered to play an important role in blood pressure regulation. Experimental structural characterization of these peptides, carried out with circular dichroism and infrared spectroscopy, showed that the angiotensins are mostly disordered but exhibit a measurable population of ordered structures at room temperature. Interestingly, these change from the polyproline helix for AngI to the left-handed helical conformation for AngII, III, and IV. Additionally, the antagonist, Ang₁₋₇, was found to contain a significant population of right handed α-helix. Anisotropy decay measurements with picosecond time resolution indicates a slower overall tumbling and a greater amplitude of internal motion in AngI and Ang₁₋₇ compared to AngII, which is consistent with a more compact and less flexible structure in AngII. To model the microscopic behavior of the peptides, 2-μs molecular dynamics simulation trajectories were generated for AngI and AngII, at 300 K using the OPLS-AA potential and SPC water. The structures sampled in the simulations mostly agree with the experimental results, showing the prevalence of disordered structures, turns and polyproline helices. The main difference is the lack of significant presence of left-handed helices in the trajectories. Additionally, the computational results predict fewer sampled conformations, tighter side-chain packing and marked increase of Phe8 solvent accessibility upon AngI truncation to AngII. Our combined approach of experiment and extensive computer simulation thus yields new information on the conformational dynamics of the angiotensins, helping provide insight into the structural basis for the potency of AngI relative to AngII.

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