THE EXPRESSION AND CELLULAR LOCALIZATION OF CC-CHEMOKINE RECEPTOR 5 (CCR5) AFTER TRAUMATIC BRAIN INJURY

Traumatic brain injury results from a primary insult and secondary events that together result in tissue injury. This primary injury occurs at the moment of impact and damage can include scalp laceration, skull fraction, cerebral contusions and lacerations as well as intracranial hemorrhage. Following the initial insult, a delayed response occurs and is characterized by hypoxia, ischemia, cerebral edema, and infection. During secondary brain injury, a series of neuroinflammatory events are triggered that can produce additional damage but may also help to protect nervous tissue from invading pathogens and help to repair the damaged tissue. Brain microglia and astrocytes become activated and migrate to the site of injury where these cells secrete immune mediators such as cytokines and chemokines. CC-chemokine receptor 5 (CCR5) is a member of the CC chemokine receptor family of seven transmembrane G protein coupled receptors. CCR5 is expressed in the immune system and is found in monocytes, leukoctyes, memory T cells, and immature dendritic cells. Upon binding to its ligands, CCR5 functions in the chemotaxis of these immune cells to the site of inflammation. In the CNS, CCR5 and its ligands are expressed in multiple cell types. In this study, I investigated whether CCR5 expression is altered in brain after traumatic brain injury. I examined the time course of CCR5 protein expression in cortex and hippocampus using quantitative western analysis of tissues from injured rat brain after mild impact injury. In addition, I also investigated the cellular localization of CCR5 before and after brain injury using confocal microscopy. I have observed that after brain injury CCR5 is upregulated in a time dependent manner in neurons of the parietal cortex and hippocampus. The absence of CCR5 expression in microglia and its delayed expression in neurons after injury suggests a role for CCR5 in neuronal survival after injury.

DIRECT INPUTS TO OFF Α AND G9 GANGLION CELLS FROM AII AMACRINE CELLS IN RABBIT RETINA

In the mammalian retina, AII amacrine cells are essential in the rod pathway for dark-adapted vision. But they also have a “day job”, to provide inhibitory inputs to certain OFF ganglion cells in photopic conditions. This is known as crossover inhibition. Physiological evidence from several different labs implies that AII amacrine cells provide direct input to certain OFF ganglion cells. However, previous EM analysis of the rabbit retina suggests that the dominant output of the AII amacrine cell in sublamina a goes to OFF cone bipolar cells (Strettoi et al., 1992). Two OFF ganglion cell types in the rabbit retina, OFF α and G9, were identified by a combination of morphological criteria such as dendritic field size, dye coupling, mosaic properties and stratification depth. The AII amacrine cells (AIIs) were labeled with an antibody against calretinin and glycine receptors were marked with an antibody against the α1 subunit. This material was analyzed by triple-label confocal microscopy. We found the lobules of AIIs made close contacts at many points along the dendrites of individual OFF α and G9 ganglion cells. At these potential synaptic sites, we also found punctate labeling for the glycine receptor α1 subunit. The presence of a post-synaptic marker such as the α1 glycine receptor at contact points between AII lobules and OFF ganglion cells supports a direct inhibitory input from AIIs. This pathway provides for crossover inhibition in the rabbit retina whereby light onset provides an inhibitory signal to OFF α and G9 ganglion cells. Thus, these two OFF ganglion cell types receive a mixed excitatory and inhibitory drive in response to light stimulation.

A Cell Biological Determination of Integrator Subunit Localization

Uridine-rich small nuclear (U snRNAs), with the exception of the U6 snRNA, are RNA polymerase II (RNAPII) transcripts. The mechanism of 3’ cleavage of snRNAs has been unknown until recently. This area was greatly advanced when 12 of the Integrator complex subunits (IntS) were purified in 2005 through their interaction with the C-terminal domain (CTD) of the large subunit (RpbI) of RNAPII. Subsequently, our lab performed a genome-wide RNAi screen that identified two more members of the complex that we have termed IntS13 and IntS14. We have determined that IntS9 and 11 mediate the 3’ cleavage of snRNAs, but the exact function of the other subunits remains unknown. However, through the use of a U7 snRNA-GFP reporter and RNAi knockdown of the Integrator subunits in Drosophila S2 cells, we have shown that all subunits are required for the proper processing of snRNAs, albeit to differing degrees. Because snRNA transcription takes place in the nucleus of the cell, it is expected that all of the Integrator subunits would exhibit nuclear localization, but the knowledge of discrete subnuclear localization (i.e. to Cajal bodies) of any of the subunits could provide important clues to the function of that subunit. In this study, we used a cell biological approach to determine the localization of the 14 Integrator subunits. We hypothesized that the majority of the subunits would be nuclear, however, a few would display distinct localization to the Cajal bodies, as this is where snRNA genes are localized and transcribed. The specific aims and results are: 1. To determine the subcellular localization of the 14 Integrator subunits. To accomplish this, mCherry and GFP tagged clones were generated for each of the 14 Drosophila and human Integrator subunits. Confocal microscopy studies revealed that the majority of the subunits were diffuse in the nucleus, however, IntS3 formed discrete subnuclear foci. Surprisingly, two of the subunits, IntS2 and 7 were observed in cytoplasmic foci. 2. To further characterize Integrator subunits with unique subcellular localizations. Colocalization studies with endogenous IntS3 and Cajal body marker, coilin, showed that these two proteins overlap, and from this we concluded that IntS3 localized to Cajal bodies. Additionally, colocalization studies with mCherry-tagged IntS2 and 7 and the P body marker, Dcp1, revealed that these proteins colocalize as well. IntS7, however, is more stable in cytoplasmic foci than Dcp1. It was also shown through RNAi knockdown of Integrator subunits, that the cytoplasmic localization of IntS2 and 7 is dependent on the expression of IntS1 and 11 in S2 cells.

Size-dependent accumulation of particles in lysosomes modulates dendritic cell function through impaired antigen degradation.

INTRODUCTION Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC) networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. METHODS Bone marrow-derived DCs (BMDCs) were exposed in vitro to 20 or 1,000 nm polystyrene (PS) particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4(+) T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. RESULTS The frequency of PS particle-positive CD11c(+)/CD11b(+) BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity of BMDCs to degrade soluble antigen, without affecting their ability to induce antigen-specific CD4(+) T-cell proliferation. Co-localization studies between PS particles and lysosomes using laser scanning confocal microscopy detected a significantly higher frequency of co-localized 20 nm particles as compared with their 1,000 nm counterparts. Neither size of PS particle caused lysosomal leakage, expression of endoplasmic reticulum stress gene markers, or changes in cytokines profiles. CONCLUSION These data indicate that although supposedly inert PS nanoparticles did not induce DC activation or alteration in CD4(+) T-cell stimulating capacity, 20 nm (but not 1,000 nm) PS particles may reduce antigen degradation through interference in the lysosomal compartment. These findings emphasize the importance of performing in-depth analysis of DC function when developing novel approaches for immune modulation with nanoparticles.

Alteration of ZnT5-mediated zinc import into the early secretory pathway affects the secretion of growth hormone from rat pituitary cells

BACKGROUND Aggregation of growth hormone (GH) required for its proper storage in granules is facilitated by zinc (Zn(2+)) transported by specific zinc transporters in and out of the regulated secretory pathway. Slc30a5 (ZnT5) was reported to have the highest gene expression among all zinc transporters in primary mouse pituitary cells while ZnT5-null mice presented with abnormal bone development and impaired growth compared to wild-type counterparts. METHODS In vitro studies performed in GH3 cells, a rat pituitary cell line that endogenously produces rat GH (rGH), included analysis of: cytoplasmic Zn(2+) pool changes after altering rSlc30a5 expression (luciferase assay), rZnT5 association with different compartments of the regulated secretory pathway (confocal microscopy), and the rGH secretion after rSlc30a5 knock-down (Western blot). RESULTS Confocal microscopy demonstrated high co-localization of rZnT5 with ER and Golgi (early secretory pathway) while siRNA-mediated knock-down of rSlc30a5 gene expression led to a significant reduction in rGH secretion. Furthermore, altered expression of rSlc30a5 (knock-down/overexpression) evoked changes in the cytoplasmic Zn(2+) pool indicating its important role in mediating Zn(2+) influx into intracellular compartments of the regulated secretory pathway. CONCLUSION Taken together, these results suggest that ZnT5 might play an important role in regulated GH secretion that is much greater than previously anticipated.

Heterologous expression from the human D-Loop in organello

We report the expression of a linear reporter construct in isolated human mitochondria. The reporter construct contained the entire human D-Loop with adjacent tRNA (MTT) genes (mt.15956-647), the human ND1 gene with an in frame GFP gene and adjacent endogenous MTT genes and heterologous rat MTT genes. Natural competence of isolated human mitochondria of HepG2 cells was used to import reporter constructs. The import efficiency of various fluorescently labelled PCR-generated import substrates in the range of 250bp up to 3.5kb was assessed by quantitative PCR and evaluated by confocal microscopy. Heterologous expression of the imported construct was confirmed at RNA level by a circular RNA (cRNA)-RT-PCR assay for the expression of tRNAs and by in organello [α-(32)P]-UTP labelling and subsequent hybridisation to reporter-specific sequences for monitoring mRNA expression. Heterologous expression of rat mitochondrial tRNA(Leu(UUR)) (rMT-TL1) was confirmed by co-/post-transcriptional trinucleotide (CCA) addition. Interestingly, the rat-specific MT-TL1 was correctly processed in isolated human mitochondria at the 3' end, but showed an aberrant 5' end processing. Correct 3' end processing of the heterologous expressed mitochondrial rat tRNA(Ser2) (MT-TS2) was detected. These findings demonstrate the feasibility of genetic manipulation of human mitochondria, providing a tool for characterisation of cis-acting elements of the human mitochondrial genome and for the study of human mitochondrial tRNA processing in organello.

Increased nuclear suppressor of cytokine signaling 1 in asthmatic bronchial epithelium suppresses rhinovirus induction of innate interferons.

BACKGROUND Rhinovirus infections are the dominant cause of asthma exacerbations, and deficient virus induction of IFN-α/β/λ in asthmatic patients is important in asthma exacerbation pathogenesis. Mechanisms causing this interferon deficiency in asthmatic patients are unknown. OBJECTIVE We sought to investigate the expression of suppressor of cytokine signaling (SOCS) 1 in tissues from asthmatic patients and its possible role in impaired virus-induced interferon induction in these patients. METHODS We assessed SOCS1 mRNA and protein levels in vitro, bronchial biopsy specimens, and mice. The role of SOCS1 was inferred by proof-of-concept studies using overexpression with reporter genes and SOCS1-deficient mice. A nuclear role of SOCS1 was shown by using bronchial biopsy staining, overexpression of mutant SOCS1 constructs, and confocal microscopy. SOCS1 levels were also correlated with asthma-related clinical outcomes. RESULTS We report induction of SOCS1 in bronchial epithelial cells (BECs) by asthma exacerbation-related cytokines and by rhinovirus infection in vitro. We found that SOCS1 was increased in vivo in bronchial epithelium and related to asthma severity. SOCS1 expression was also increased in primary BECs from asthmatic patients ex vivo and was related to interferon deficiency and increased viral replication. In primary human epithelium, mouse lung macrophages, and SOCS1-deficient mice, SOCS1 suppressed rhinovirus induction of interferons. Suppression of virus-induced interferon levels was dependent on SOCS1 nuclear translocation but independent of proteasomal degradation of transcription factors. Nuclear SOCS1 levels were also increased in BECs from asthmatic patients. CONCLUSION We describe a novel mechanism explaining interferon deficiency in asthmatic patients through a novel nuclear function of SOCS1 and identify SOCS1 as an important therapeutic target for asthma exacerbations.

Materno-fetal nutrient transfer across primary human trophoblast layer.

MATERNO-FETAL NUTRIENT TRANSFER ACROSS PRIMARY HUMAN TROPHOBLAST MONOLAYER Objectives: Polarized trophoblasts represent the transport and metabolic barrier between the maternal and fetal circulation. Currently human placental nutrient transfer in vitro is mainly investigated unidirectionallyon cultured primary trophoblasts, or bidirectionally on the Transwell® system using BeWo cells treated with forskolin. As forskolin can induce various gene alterations (e.g. cAMP response element genes), we aimed to establish a physiological primary trophoblast model for materno-fetal nutrient exchange studies without forskolin application. Methods: Human term cytotrophoblasts were isolated by enzymatic digestion and Percoll® gradient separation. The purity of the primary cells was assessed by flow cytometry using the trophoblast-specific marker cytokeratin-7. After screening different coating matrices, we optimized the growth conditions for the primary cytotrophoblasts on Transwell/ inserts. The morphology of 5 days cultured trophoblasts was determined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane makers were visualized using confocal microscopy. Additionally transport studies were performed on the polarized trophoblasts in the Transwell® system. Results: During 5 days culture, the trophoblasts (>90% purity) developed a modest trans-epithelial electrical resistance (TEER) and a sizedependent apparent permeability coefficient (Papp) to fluorescently labeled compounds (MW ~400-70’000D). SEM analyses confirmed a confluent trophoblast layer with numerous microvilli at day six, and TEM revealed a monolayer with tight junctions. Immunocytochemistry on the confluent trophoblasts showed positivity for the cell-cell adhesion molecule E-cadherin, the tight junction protein ZO-1, and the membrane proteins ABCA1 and Na+/K+-ATPase. Vectorial glucose and cholesterol transport studies confirmed functionality of the cultured trophoblast barrier. Conclusion: Evidence from cell morphology, biophysical parameters and cell marker expressions indicate the successful and reproducible establishment of a primary trophoblast monolayer model suitable for transport studies. Application of this model to pathological trophoblasts will help to better understand the mechanism underlying gestational diseases, and to define the consequences of placental pathology on materno-fetal nutrient transport.

Dendritic spine morphology determines membrane-associated protein exchange between dendritic shafts and spine heads.

The purpose of this study was to examine whether variability in the shape of dendritic spines affects protein movement within the plasma membrane. Using a combination of confocal microscopy and the fluorescence loss in photobleaching technique in living hippocampal CA1 pyramidal neurons expressing membrane-linked GFP, we observed a clear correlation between spine shape parameters and the diffusion and compartmentalization of membrane-associated proteins. The kinetics of membrane-linked GFP exchange between the dendritic shaft and the spine head compartment were slower in dendritic spines with long necks and/or large heads than in those with short necks and/or small heads. Furthermore, when the spine area was reduced by eliciting epileptiform activity, the kinetics of protein exchange between the spine compartments exhibited a concomitant decrease. As synaptic plasticity is considered to involve the dynamic flux by lateral diffusion of membrane-bound proteins into and out of the synapse, our data suggest that spine shape represents an important parameter in the susceptibility of synapses to undergo plastic change.

Active eosinophilic esophagitis is characterized by epithelial barrier defects and eosinophil extracellular trap formation.

BACKGROUND Eosinophilic esophagitis (EoE) exhibits esophageal dysfunction owing to an eosinophil-predominant inflammation. Activated eosinophils generate eosinophil extracellular traps (EETs) able to kill bacteria. There is evidence of an impaired barrier function in EoE that might allow pathogens to invade the esophagus. This study aimed to investigate the presence and distribution of EETs in esophageal tissues from EoE patients and their association with possible epithelial barrier defects. METHODS Anonymized tissue samples from 18 patients with active EoE were analyzed. The presence of DNA nets associated with eosinophil granule proteins forming EETs and the expression of filaggrin, the protease inhibitor lympho-epithelial Kazal-type-related inhibitor (LEKTI), antimicrobial peptides, and cytokines were evaluated by confocal microscopy following immune fluorescence staining techniques. RESULTS Eosinophil extracellular trap formation occurred frequently and was detected in all EoE samples correlating with the numbers of infiltrating eosinophils. While the expression of both filaggrin and LEKTI was reduced, epithelial antimicrobial peptides (human beta-defensin-2, human beta-defensin-3, cathelicidin LL-37, psoriasin) and cytokines (TSLP, IL-25, IL-32, IL-33) were elevated in EoE as compared to normal esophageal tissues. There was a significant correlation between EET formation and TSLP expression (P = 0.02) as well as psoriasin expression (P = 0.016). On the other hand, a significant negative correlation was found between EET formation and LEKTI expression (P = 0.016). CONCLUSION Active EoE exhibits the presence of EETs. Indications of epithelial barrier defects in association with epithelial cytokines are also present which may have contributed to the activation of eosinophils. The formation of EETs could serve as a firewall against the invasion of pathogens.

Preclinical evaluation of proteasome inhibition for the treatment of prostate cancer

Prostatic carcinoma is the most prevalent cancer detected in men. Bortezomib is the first proteasome inhibitor to undergo clinical trials for several forms of cancer. Although we know this class of agent preferentially kills cancer cells, our knowledge of proteasome inhibition mechanisms of induced death is far from complete. We investigated the effects of bortezomib on the LNCaP-Pro5 (Pro5) and PC-3-Pro4 (Pro4) human prostatic adenocarcinoma cells lines. We showed a reduction in proliferation and an increase in DNA fragmentation, caspase 3 activity, and cell surface phosphatidyl serine exposure. The bortezomib-treated tumors from both cell lines were dramatically reduced, and apoptosis was induced. There was also a reduction in proliferation in the treated tumors from both cells lines. We looked at changes in the levels of the proangiogenic factors VEGF, IL-8 and bFGF in vitro and in vivo. Although there was a reduction in the levels of VEGF produced by the Pro5 cell line and tumor due to bortezomib, no similar observations were made for the other angiogenic factors or in the Pro4 cells. We investigated the effects of bortezomib on p53 in the Pro5 cell line. Bortezomib induced strong stabilization of p53. It did not promote phosphorylation on serines 15 and 24 and p53 remained bound to its inhibitor, mdm2. Nonetheless, confocal microscopy revealed that bortezomib stimulated p53 translocation to the nucleus and enhanced p53 DNA binding, accumulation of p53-dependant transcripts, and activation of a p53-responsive reporter gene. Furthermore, stable transfectants of LNCaP-Pro5 expressing the p53 inhibitor, HPV-E6, displayed reduced bortezomib-induced p53 activation and cell death. Our data shows bortezomib to induce antitumor effects in the human Pro4 and Pro5 prostatic adenocarcinoma cell lines by the direct induction of apoptosis. The drug also causes a reduction in cell proliferation and mean vessel density while modulating the secretion of proangiogenic factors. Although we show that proteasome inhibition stimulates p53 activation via a novel mechanism in Pro5 cells, it is also toxic to p53 null cells as is seen in the Pro4 line. ^

The role of sensory neuron outgrowth in long-term sensitization of the tail-elicited tail-siphon withdrawal reflex of Aplysia

Neuronal outgrowth has been proposed in many systems as a mechanism underlying memory storage. For example, sensory neuron outgrowth is widely accepted as an underlying mechanism of long-term sensitization of defensive withdrawal reflexes in Aplysia. The hypothesis is that learning leads to outgrowth and consequently to the formation of new synapses, which in turn strengthen the neural circuit underlying the behavior. However, key experiments to test this hypothesis have never been performed. ^ Four days of sensitization training leads to outgrowth of siphon sensory neurons mediating the siphon-gill withdrawal response in Aplysia . We found that a similar training protocol produced robust outgrowth in tail sensory neurons mediating the tail siphon withdrawal reflex. In contrast, 1 day of training, which effectively induces long-term behavioral sensitization and synaptic facilitation, was not associated with neuronal outgrowth. Further examination of the effect of behavioral training protocols on sensory neuron outgrowth indicated that this structural modification is associated only with the most persistent forms of sensitization, and that the induction of these changes is dependent on the spacing of the training trials over multiple days. Therefore, we suggest that neuronal outgrowth is not a universal mechanism underlying long-term sensitization, but is involved only in the most persistent forms of the memory. ^ Sensory neuron outgrowth presumably contributes to long-term sensitization through formation of new synapses with follower motor neurons, but this hypothesis has never been directly tested. The contribution of outgrowth to long-term sensitization was assessed using confocal microscopy to examine sites of contact between physiologically connected pairs of sensory and motor neurons. Following 4 days of training, the strength of both the behavior and sensorimotor synapse and the number of appositions with follower neurons was enhanced only on the trained side of the animal. In contrast, outgrowth was induced on both sides of the animal, indicating that although sensory neuron outgrowth does appear to contribute to sensitization through the formation of new synapses, outgrowth alone is not sufficient to account for the effects of sensitization. This indicates that key regulatory steps are downstream from outgrowth, possibly in the targeting of new processes and activation of new synapses. ^

Diversity of neuronal connexins in the mammalian retina

Many neurons in the mammalian retina are electrically coupled by intercellular channels or gap junctions, which are assembled from a family of proteins called connexins. Numerous studies indicate that gap junctions differ in properties such as conductance and tracer permeability. For example, A-type horizontal cell gap junctions are permeable to Lucifer Yellow, but B-type horizontal cell gap junctions are not. This suggests the two cell types express different connexins. My hypothesis is that multiple neuronal connexins are expressed in the mammalian retina in a cell type specific manner. Immunohistochemical techniques and confocal microscopy were used to localize certain connexins within well-defined neuronal circuits. The results of this study can be summarized as follows: AII amacrine cells, which receive direct input from rod bipolar cells, are well-coupled to neighboring AIIs. In addition, AII amacrine cells also form gap junctions with ON cone bipolar cells. This is a complex heterocellular network. In both rabbit and primate retina, connexin36 occurs at dendritic crossings in the AII matrix as well as between AIIs and ON cone bipolar cells. Coupling in the AII network is thought to reduce noise in the rod pathway while AII/bipolar gap junctions are required for the transmission of rod signals to ON ganglion cells. In the outer plexiform layer, connexin36 forms gap junctions between cones and between rods and cones via cone telodendria. Cone to cone coupling is thought to reduce noise and is partly color selective. Rod to cone coupling forms an alternative rod pathway thought to operate at intermediate light intensity. A-type horizontal cells in the rabbit retina are strongly coupled via massive low resistance gap junctions composed from Cx50. Coupling dramatically extends the receptive field of horizontal cells and the modulation of coupling is thought to change the strength of the feedback signal from horizontal cells to cones. Finally, there are other coupled networks, such as B-type horizontal cells and S1/S2 amacrine cells, which do not use either connexin36 or Cx50. These results confirm the hypothesis that multiple neuronal connexins are expressed in the mammalian retina and these connexins are localized to particular retinal circuits. ^

Why does low dose morphine augment fentanyl analgesia?

Opioids dominate the field of pain management because of their ability to provide analgesia in many medical circumstances. However, side effects including respiratory depression, constipation, tolerance, physical dependence, and the risk of addiction limit their clinical utility. Fear of these side effects results in the under-treatment of acute pain. For many years, research has focused on ways to improve the therapeutic index (the ratio of desirable analgesic effects to undesirable side effects) of opioids. One strategy, combining opioid agonists that bind to different opioid receptor types, may prove successful.^ We discovered that subcutaneous co-administration of a moderately analgesic dose of the mu-opioid receptor (MOR) selective agonist fentanyl (20μg/kg) with subanalgesic doses of the less MOR-specific agonist morphine (100ng/kg-100μg/kg), augmented acute fentanyl analgesia in rats. Parallel [35S]GTPγS binding studies using naïve rat substantia gelatinosa membrane treated with fentanyl (4μM) and morphine (1nM-1pM) demonstrated a 2-fold increase in total G-protein activation. This correlation between morphine-induced augmentation of fentanyl analgesia and G-protein activation led to our proposal that interactions between MORs and DORs underlie opioid-induced augmentation. We discovered that morphine-induced augmentation of fentanyl analgesia and G-protein activity was mediated by DORs. Adding the DOR-selective antagonist naltrindole (200ng/kg, 40nM) at doses that did not alter the analgesic or G-protein activation of fentanyl, blocked increases in analgesia and G-protein activation induced by fentanyl/morphine combinations. Equivalent doses of the MOR-selective antagonist cyprodime (20ng/kg, 4nM) did not block augmentation. Substitution of the DOR-selective agonist SNC80 for morphine yielded similar results, further supporting our conclusion that interactions between MORs and DORs are responsible for morphine-induced augmentation of fentanyl analgesia and G-protein activation. Confocal microscopy of rat substantia gelatinosa showed that changes in the rate of opioid receptor internalization did not account for these effects.^ In conclusion, fentanyl analgesia augmentation by subanalgesic morphine is mediated by increased G-protein activation resulting from functional interactions between MORs and DORs, not changes in MOR internalization. Additional animal and clinical studies are needed to determine whether side effect incidence changes following opioid co-administration. If side effect incidence decreases or remains unchanged, these findings could have important implications for clinical pain treatment. ^

EVIDENCE OF HUMAN ENDOGENOUS RETROVIRUS K INVOLVEMENT IN HUMAN CANCER

Many lines of clinical and experimental evidence indicate a viral role in carcinogenesis (1-6). Our access to patient plasma, serum, and tissue samples from invasive breast cancer (N=19), ductal carcinoma in situ (N=13), malignant ovarian cancer (N=12), and benign ovarian tumors (N=9), via IRB-approved and informed consent protocols through M.D. Anderson Cancer Center, as well as normal donor plasmas purchased from Gulf Coast Regional Blood Center (N=6), has allowed us to survey primary patient blood and tissue samples, healthy donor blood from the general population, as well as commercially available human cell lines for the presence of human endogenous retrovirus K (HERV-K) Env viral RNA (vRNA), protein, and viral particles. We hypothesize that HERV-K proteins are tumor-associated antigens and as such can be profiled and targeted in patients for diagnostic and therapeutic purposes. To test this hypothesis, we employed isopycnic ultracentrifugation, a microplate-based reverse transcriptase enzyme activity assay, reverse transcription – polymerase chain reaction (RT-PCR), cDNA sequencing, SDS-PAGE and western blotting, immunofluorescent staining, confocal microscopy, and transmission electron microscopy to evaluate v HERV-K activation in cancer. Data from large numbers of patients tested by reverse transcriptase activity assay were analyzed statistically by t-test to determine the potential use of this assay as a diagnostic tool for cancer. Significant reverse transcriptase enzyme activity was detected in 75% of ovarian cancer patients, 53.8% of ductal carcinoma in situ patient, and 42.1% of invasive breast cancer patient samples. Only 11.1% of benign ovarian patient and 16.7% of normal donor samples tested positive. HERV-K Env vRNA, or Env SU were detected in the majority of cancer types screened, as demonstrated by the results shown herein, and were largely absent in normal controls. These findings support our hypothesis that the presence of HERV-K in patient blood circulation is an indicator of cancer or pre-malignancy in vivo, that the presence of HERV-K Env on tumor cell surfaces is indicative of malignant phenotype, and that HERV-K Env is a tumor-associated antigen useful not only as a diagnostic screening tool to predict patient disease status, but also as an exploitable therapeutic target for various novel antibody-based immunotherapies.