Subtype-specific Antibodies Research Articles

Synergistic Roles for G-protein γ3 and γ7 Subtypes in Seizure Susceptibility as Revealed in Double Knock-out Mice

The functions of different G-protein αβγ subunit combinations are traditionally ascribed to their various α components. However, the discovery of similarly diverse γ subtypes raises the possibility that they may also contribute to specificity. To test this possibility, we used a gene targeting approach to determine whether the closely related γ(3) and γ(7) subunits can perform functionally interchangeable roles in mice. In contrast to single knock-out mice that show normal survival, Gng3(-/-)Gng7(-/-) double knock-out mice display a progressive seizure disorder that dramatically reduces their median life span to only 75 days. Biochemical analyses reveal that the severe phenotype is not due to redundant roles for the two γ subunits in the same signaling pathway but rather is attributed to their unique actions in different signaling pathways. The results suggest that the γ(3) subunit is a component of a G(i/o) protein that is required for γ-aminobutyric acid, type B, receptor-regulated neuronal excitability, whereas the γ(7) subunit is a component of a G(olf) protein that is responsible for A(2A) adenosine or D(1) dopamine receptor-induced neuro-protective response. The development of this mouse model offers a novel experimental framework for exploring how signaling pathways integrate to produce normal brain function and how their combined dysfunction leads to spontaneous seizures and premature death. The results underscore the critical role of the γ subunit in this process.

Development of H5 subtype-specific monoclonal antibodies (MAb) and MAb-based assays for rapid detection of H5 avian influenza

Avian influenza (AI) virology surveillance is the most important method to monitor AI virus (AIV) in poultry so as to effectively prevent and control AI outbreaks. Monoclonal antibodies (MAb)-based assays are highly sensitive and specific for AIV detection, and much practical and economic for test-in-field or onsite. Many such assays have been developed and are still in developing since the H5N1 highly pathogenic AI (HPAI) outbreaks occurred in South East Asia in 2003. A MAb-based dot-enzyme-linked immunosorbent assay (ELISA) has been developed in our lab during late 1990s and early 2000s. Meanwhile, AIV H7 and H5 subtype specific-MAbs have been successfully developed in our laboratory to enhance the Dot-ELISA and other MAb-based assays for AIV detection. Production and purification of the H7 and H5 MAbs were made to provide essential reagents for Dot-ELISA and other immunoassays, and the current development of a novel Biosensor technique for rapid detection of AIV from clinical and field specimens.

Aripiprazole for the Treatment of Involuntary Movement Symptoms in Patients With Major Depressive Disorder

Aripiprazole for the Treatment of Involuntary Movement Symptoms in Patients With Major Depressive Disorder

Monoclonal Antibodies against the Human Somatostatin Receptor Subtypes 1–5: Development and Immunohistochemical Application in Neuroendocrine Tumors

Background: Activation of somatostatin receptors (sstr1–5) by somatostatin and its analogues exerts an inhibitory effect on hormone secretion and provides the basis for the treatment of a range of endocrine diseases such as acromegaly, Cushing’s disease and neuroendocrine tumors (NET). The lack of well-characterized commercially available sstr subtype-specific antibodies prevents routine identification of the sstr expression profile in patients. Methods: We generated and characterized new mouse monoclonal antibodies (mAbs) targeting the five human sstr subtypes using ELISA and immunohistochemistry, and tested their suitability in formalin-fixed and paraffin-embedded (FFPE) human tissues and archival samples of normal pancreatic tissue and NET. Results: All mAbs were highly specific with no cross-reactivity. The sstr1–5 immunoreactivity in gastrointestinal NET (n = 67) was correlated with clinicopathologic data. With the exception of sstr3, NET were highly positive for all receptor subtypes (42, 63, 6, 32 and 65% of tumors were positive for sstr1, sstr2a, sstr3, sstr4 and sstr5, respectively). sstr1, sstr2a and sstr5 were present at the plasma membrane and in the cytoplasm of tumor cells, whereas sstr3 and sstr4 were almost exclusively cytoplasmic. Immunoreactivity of sstr1, sstr2a and sstr4 tended to decrease as tumor aggressiveness increased. sstr5 showed an opposite pattern, with higher staining in well-differentiated carcinomas compared with well-differentiated tumors. sstr5 immunoreactivity was correlated with the presence of metastases and angioinvasion, suggesting a possible association with more aggressive behavior. Conclusion: Determination of the sstr1–5 by immunohistochemistry using subtype-specific mAbs is feasible in FFPE tissue and may provide a tool for routine clinical practice.

A Novel Murine Model of Pregnancy and Transfusion Induced Anti-Kell RBC Alloimmunization

Abstract 715 Introduction:Pregnancy and transfusion induced RBC alloimmunization can be clinically significant, leading to difficulties with future transfusions and also leading to hemolytic disease of the fetus and newborn (HDFN). Maternal anti-Kell alloimmunization is one of the most common causes of non ABO-mediated HDFN. To date, there are few treatments to prevent HDFN due to RBC antibodies outside of the Rh(D) family, and there are few existing animal models of pregnancy induced RBC alloimmunization. Herein, we describe a novel murine model in which Kell RBC alloimmunization occurs following pregnancy or transfusion. Materials and Methods:Mice with RBC specific expression of the K2 allele of the human Kell glycoprotein (subsequently referred to as “K2” mice) were generated utilizing constructs containing the human K2 sequence expressed by beta-globin regulatory elements. For the transfusion induced alloimmunization experiments, C57BL/6 female recipients were transfused 3 times with K2 RBCs in the presence of poly (I:C), with anti-Kell glycoprotein antibodies measured after each transfusion by flow cytometric crossmatch using K2 targets and subtype specific antibodies. For the pregnancy induced alloimmunization experiments, control naïve C57BL/6 females were mated with K2 males three times; females immunized following transfusion were mated with K2 males as well. After the final pregnancy, anti-Kell glycoprotein antibodies were also measured by flow cytometric. Number and percentage of K2 positive pups were noted in a subset of pregnancies, and deceased pups were genotyped by PCR. Results:Anti-Kell glycoprotein antibodies were detectable in C57BL/6 recipients following either K2 RBC transfusion or following repeat pregnancies with K2 positive fetuses. In 5 experiments, 30/30 transfused mice had anti-Kell antibody titers that increased with subsequent transfusion, with IgM, IgG1, IgG2b, IgG2c, and IgG3 anti-Kell being detectable. After 2–3 pregnancies with K2 mates, 11/12 C57BL/6 females developed anti-Kell glycoprotein antibodies, with IgM and all IgG anti-Kell subtypes being present. Regardless of the mechanism of immunization (e.g. transfusion or prior pregnancy), live births and pups surviving beyond day of life #2 were lower in Kell immunized mothers compared with control non-immunized mothers (approximately 2–4 pups compared to 6–10 pups). At least one K2 stillborn pup had severe edema resembling hydrops. Discussion:This is the first animal model of Kell RBC alloimmunization, in which anti-Kell can be generated either through prior pregnancy or transfusion and appears to be detrimental to pups. This model mimics that of pregnancy or transfusion induced anti-Rh(D), in that the described antigen (K2) is largely foreign to the C57BL/6 recipients (who lack human K1 or K2 altogether). Ongoing studies are investigating whether a particular method of immunization (e.g. pregnancy vs transfusion) is more detrimental to fetuses and pups. As expected, the anti-Kell antibody appears more detrimental to K2 positive than negative fetuses, with immunized mothers having smaller litters of predominantly K2 negative fetuses; however, K2 negative pups may also have higher rates of demise following birth to immunized compared to non-immunized mothers. In sum, this model will allow for further investigation of pregnancy and transfusion induced anti-Kell RBC alloimmunization, and may also serve to increase the understanding of the pathogenesis and prevention of HDFN. Disclosures:Zimring:Immucor: Funds from an Immucor sponsored project (unrelated to the current project) were utilized in part to generate the KEL transgenic animals.

Evaluation study of a portable impedance biosensor for detection of avian influenza virus

Current methods for detection of avian influenza virus (AIV) based on virus culture and RT-PCR are well established, but they are either time consuming or require specialized laboratory facilities and highly trained technicians. A simple, rapid, robust, and reliable test, suitable for use in the field or at the patient's bedside, is urgently needed. In this study, the performance of a newly developed portable impedance biosensor was evaluated by comparison with real-time reverse transcriptase PCR (rRT-PCR) and virus culture for detection of AIV in tracheal and cloacal swab samples collected from experimentally H5N2 AIV infected chickens. The impedance biosensor system was based on a combination of magnetic nanobeads, which were coated with AIV subtype-specific antibody for capture (separation and concentration) of a target virus, and a microfluidic chip with an interdigitated array microelectrode for transfer and detection of target virus, and impedance measurement of the bio-nanobeads and AI virus complexes in a buffer solution. A comparison of results obtained from 59 swab samples using virus culture, impedance biosensor and rRT-PCR methods showed that the impedance biosensor technique was comparable in sensitivity and specificity to rRT-PCR. Detection time for the impedance biosensor is less than 1h.

Localization of Cadm2a and Cadm3 proteins during development of the zebrafish nervous system

Members of the Cadm/SynCAM/Necl/IGSF/TSLC family of cell adhesion molecules are known to have diverse functions during development of the nervous system, but information regarding their role during central nervous system (CNS) development in vivo is scarce. The rapid development of a relatively simple nervous system in larval zebrafish makes them a highly tractable model organism for studying gene function during nervous system development. An essential prerequisite for functional studies is a description of protein localization. To address this we have generated subtype-specific antibodies to two members of the zebrafish cell adhesion molecule family: cadm2a and cadm3. Using these novel antibodies we show that cadm3 and cadm2a are expressed throughout the nervous system of larval stage zebrafish. Particularly striking, and largely nonoverlapping expression of cadm2a and cadm3 is observed in the developing retina and spinal cord. Using in vitro binding assays we show that cadm2a and cadm3 bind heterophilically and preferentially to cadm1 and cadm4, respectively. These binding preferences are very similar to those seen for tetrapod Cadms but our study of protein localization suggests novel and diverse functions of cadms during nervous system development.

Cholinergic microvillous cells in the mouse main olfactory epithelium and effect of acetylcholine on olfactory sensory neurons and supporting cells

The mammalian olfactory epithelium is made up of ciliated olfactory sensory neurons (OSNs), supporting cells, basal cells, and microvillous cells. Previously, we reported that a population of nonneuronal microvillous cells expresses transient receptor potential channel M5 (TRPM5). Using transgenic mice and immunocytochemical labeling, we identify that these cells are cholinergic, expressing the signature markers of choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter. This result suggests that acetylcholine (ACh) can be synthesized and released locally to modulate activities of neighboring supporting cells and OSNs. In Ca(2+) imaging experiments, ACh induced increases in intracellular Ca(2+) levels in 78% of isolated supporting cells tested in a concentration-dependent manner. Atropine, a muscarinic ACh receptor (mAChR) antagonist suppressed the ACh responses. In contrast, ACh did not induce or potentiate Ca(2+) increases in OSNs. Instead ACh suppressed the Ca(2+) increases induced by the adenylyl cyclase activator forskolin in some OSNs. Supporting these results, we found differential expression of mAChR subtypes in supporting cells and OSNs using subtype-specific antibodies against M(1) through M(5) mAChRs. Furthermore, we found that various chemicals, bacterial lysate, and cold saline induced Ca(2+) increases in TRPM5/ChAT-expressing microvillous cells. Taken together, our data suggest that TRPM5/ChAT-expressing microvillous cells react to certain chemical or thermal stimuli and release ACh to modulate activities of neighboring supporting cells and OSNs via mAChRs. Our studies reveal an intrinsic and potentially potent mechanism linking external stimulation to cholinergic modulation of activities in the olfactory epithelium.

Broad spectrum reactivity versus subtype specificity—Trade-offs in serodiagnosis of influenza A virus infections by competitive ELISA

Avian influenza viruses (AIVs) of the H5 and H7 subtypes can cause substantial economic losses in the poultry industry and are a potential threat to public health. Serosurveillance of poultry populations is an important monitoring tool and can also be used for control of vaccination campaigns. The purpose of this study was to develop broadly reactive, yet subtype-specific competitive ELISAs (cELISAs) for the specific detection of antibodies to the notifiable AIV subtypes H5 and H7 as an alternative to the gold standard haemagglutination inhibition assay (HI). Broadly reacting monoclonal competitor antibodies (mAbs) and genetically engineered subtype H5 or H7 haemagglutinin antigen, expressed and in vivo biotinylated in insect cells, were used to develop the cELISAs. Sera from galliform species and water fowl ( n = 793) were used to evaluate the performance characteristics of the cELISAs. For the H5 specific cELISA, 98.1% test sensitivity and 91.5% test specificity (97.7% and 90.2% for galliforms; 98.9% and 92.6% for waterfowl), and for the H7 cELISA 97.3% sensitivity and 91.8% specificity (95.3% and 98.9% for galliforms; 100% and 82.7% for waterfowl) were reached when compared to HI. The use of competitor mAbs with broad spectrum reactivity within an AIV haemagglutinin subtype allowed for homogenous detection with high sensitivity of subtype-specific antibodies induced by antigenically widely distinct isolates including antigenic drift variants. However, a trade-off regarding sensitivity versus nonspecific detection of interfering antibodies induced by phylo- and antigenically closely related subtypes, e.g., H5 versus H2 and H7 versus H15, must be considered. The observed intersubtype antibody cross-reactivity remains a disturbance variable in AIV subtype-specific serodiagnosis which negatively affects specificity.

Mechanism mediating oligomeric Aβ clearance by naïve primary microglia

The accumulation of soluble oligomeric amyloid-β peptide (oAβ) proceeds the formation of senile plaques and contributes to synaptic and memory deficits in Alzheimer's disease (AD). The mechanism of mediating microglial oAβ clearance remains unclear and thought to occur via scavenger receptors (SRs) in microglia. SRs respond to their ligands in a subtype-specific manner. Therefore, we sought to identify the specific subtypes of SRs that mediate oAβ internalization and proteases that degrade oAβ species in naïve primary microglia. The component of oAβ species were characterized by western blot analysis, analytical ultracentrifugation analysis, and atomic force microscopy. The oAβ species remained soluble in the medium and microglial lysates during incubation at 37°C. SR-A, but not CD36, mediated oAβ internalization in microglia as suggested by the use of subtype-specific neutralizing antibodies and small interfering RNAs (siRNAs). Immunoprecipitation analysis showed that oAβ interacted with SR-A on the plasma membrane. After internalization, over 40% of oAβ vesicles were trafficked toward lysosomes and degraded by cysteine proteases, including cathepsin B. The inhibitors of proteasome, neprilysin, matrix metalloproteinases, and insulin degrading enzyme failed to protect internalized oAβ from degradation. Our study suggests that SR-A and lysosomal cathepsin B are critical in microglial oAβ clearance, providing insight into how microglia are involved in the clearance of oAβ and their roles in the early stages of AD.

Specific subtyping of influenza A virus using a recombinant hemagglutinin protein expressed in baculovirus

Influenza A viruses are subtyped according to antigen characterization of hemagglutinin (HA) and neuraminidase surface glycoproteins. The hemagglutination inhibition (HI) assay using reference antiserum is currently applied to serologic screening of subtype-specific antibodies in sera. The reference antiserum is made by injecting chickens with live or inactivated whole virus preparations. Nonspecific inhibitors of antisera prepared by the conventional method may affect the specificity of HI assay. In this study, highly pure recombinant proteins generated using baculovirus expression vector system based on full-length of HA (HAF) and antigenic region of HA(1) genes of H9 subtype, and also inactivated whole virus were used to immunization of chickens. Measurable antibody titers were present for treated birds after 3 weeks and generally increased after each boost. The performance of the prepared antisera was evaluated by testing a panel of known standard strains of influenza virus representing five HA subtypes. Relative to the conventional method using whole virus immunization and recombinant HAF protein, the antiserum prepared by recombinant HA(1) had a specificity of 100% for all tested subtypes. The antiserum prepared by expression of HA1 protein in baculovirus has the potential for rapid and specific HA subtyping of influenza viruses without producing antibodies specific to other viral proteins.

Characterisation of P2X receptors expressed in rat pulmonary arteries

Previous studies indicated that a P2X receptor other than the P2X1 subtype might be present in rat large, but not small pulmonary arteries. The aim here was to characterise further these P2X receptors. Isometric tension was recorded from rat isolated small (i.d. 250–500 μm) and large pulmonary artery (i.d. 1–1.5 mm) rings mounted on a wire myograph. In both tissues the P2X receptor agonist α,β-meATP evoked rapidly-developing contractions that were inhibited by the P2X antagonists NF449, PPADS and suramin in a concentration-dependent manner and eventually abolished by each. The rank order of the potency in both tissues was NF449 > PPADS = suramin. For each antagonist there was no significant difference between its potency in the small and large pulmonary arteries. Prolonged administration of a high concentration of α,β-meATP induced complete desensitisation in both tissues. RT-PCR followed by PCR with specific oligonucleotide primers, identified mRNA for all seven P2X subunits. Subtype-specific antibodies showed strong, punctate P2X1 receptor-like immunoreactivity in the majority of cells and faint, punctate staining with the anti-P2X2 and anti-P2X4 antibodies, whilst P2X5-like immunoreactivity was barely detectable and no P2X3, P2X6, and P2X7 receptor-like immunoreactivity was seen. No differences in P2X mRNA and protein expression were seen between small and large pulmonary arteries. In conclusion, the pharmacological properties and mRNA and protein expression profiles of P2X receptors in rat small and large pulmonary arteries are very similar. Thus P2X1 appears to be the predominant P2X subunit functionally expressed in smooth muscle cells of rat small and large pulmonary arteries.

Distribution of D1 and D5 dopamine receptors in the primate nucleus accumbens

The D1 family of dopamine receptors (D1R) play a critical role in modulating reward in the nucleus accumbens (NAc). A better understanding of how D1Rs modulate NAc function must take into account the contributions of the two D1R subtypes, D1 and D5. In order to determine how these two subtypes contribute to dopamine's actions in the NAc, we utilized subtype specific antibodies and immunoelectron microscopy to quantitatively determine the localization of D1 and D5 in the neuropil of the primate NAc. We found that D1 was more commonly found in dendritic shafts and spines, while D5 was more commonly found in axon terminals, preterminal axons and glial processes. However, D5 is well positioned to play an important role in postsynaptic modulation of inputs onto NAc medium spiny neurons. Approximately one third of spines contained D1 and one quarter contained D5, and as we have previously observed in the prefrontal cortex (PFC) and amygdala, these receptors overlapped extensively in dendritic spines. Similarly, we found overlap of the two D1R in axon terminals in the NAc; however, here D5 labeled the larger population of terminals and D1 was found in a subpopulation of D5 containing terminals. Given the higher affinity of D5 for dopamine, this suggest that presynaptic modulation of inputs by dopamine may be more easily evoked than in PFC where D1 is the dominate presynaptic receptor. Finally, we investigated differences between the NAc and the dorsal striatum. We found that in the caudate half of dendritic spines contain D1, significantly more than in the NAc. This suggests differences in how receptor is translated and distributed in D1 mRNA expressing medium spiny neurons in the NAc and caudate.

Regional differences in nerve terminal Na+ channel subtype expression and Na+ channel‐dependent glutamate and GABA release in rat CNS

We tested the hypothesis that expression of pre-synaptic voltage-gated sodium channel (Na(v)) subtypes coupled to neurotransmitter release differs between transmitter types and CNS regions in a nerve terminal-specific manner. Na(v) coupling to transmitter release was determined by measuring the sensitivity of 4-aminopyridine (4AP)-evoked [(3)H]glutamate and [(14)C]GABA release to the specific Na(v) blocker tetrodotoxin (TTX) for nerve terminals isolated from rat cerebral cortex, hippocampus, striatum and spinal cord. Expression of various Na(v) subtypes was measured by immunoblotting using subtype-specific antibodies. Potencies of TTX for inhibition of glutamate and GABA release varied between CNS regions. However, the efficacies of TTX for inhibition of 4AP-evoked glutamate release were greater than for inhibition of GABA release in all regions except spinal cord. The relative nerve terminal expression of total Na(v) subtypes as well as of specific subtypes varied considerably between CNS regions. The region-specific potencies of TTX for inhibition of 4AP-evoked glutamate release correlated with greater relative expression of total nerve terminal Na(v) and Na(v)1.2. Nerve terminal-specific differences in the expression of specific Na(v) subtypes contribute to transmitter-specific and regional differences in pharmacological sensitivities of transmitter release.

Notch it up to the antibodies

Genentech and Exelixis researchers and a separate team at Aveo Pharmaceuticals, have designed Notch receptor subtype-specific antibodies, which both groups hope will improve safety compared with that for γ-secretase inhibitors and other molecules that target all subtypes. The companies are now determining the optimal cancers for targeting.

A cross-sectional survey of influenza A infection, and management practices in small rural backyard poultry flocks in two regions of New Zealand

AIMS: To obtain baseline data on the management of small non-commercial backyard poultry flocks, in two rural regions of New Zealand, to investigate potential transmission pathways for avian influenza (AI), and to investigate the presence of AI in these flocks. METHODS: During August–October 2006 a questionnaire was sent to 105 farms in the Bay of Plenty and Wairarapa with poultry flocks comprising fewer than 50 chickens, located near wetlands where AI virus had been detected previously in wild ducks. Information was collected on the number and species of poultry reared, opportunities for interaction between wild birds and poultry, farm biosecurity measures, and health status of poultry. Between September and November 2006, blood and tracheal/cloacal swabs were collected from poultry on a subset of 12 high-risk farms in each location. Influenza A-specific antibodies in sera were assayed using ELISA, and positive sera were further tested for the presence of H5 and H7 subtype-specific antibodies, using haemagglutination inhibition (HI) assay. The presence of influenza A virus in swabs was detected using realtime reverse transcriptase-PCR (RRT-PCR). RESULTS: Returned questionnaires were received from 54 farms. Overall, 80% had only chickens, 13% chickens and ducks, and 7% had chickens and other galliform species. Nearly all (96%) kept backyard chickens for personal consumption of eggs, with a small proportion (19%) preparing birds for the table. On surveyed farms wild waterfowl were seen on pastures (70%) and/or farm waterways (46%). Waterfowl were recorded as visiting areas where domestic birds were kept on 31% of farms. Bird litter and manure were composted (94%) or buried (6%) on-farm, as were most (82%) dead birds. During the targeted cross-sectional survey of 24 farms clinical disease was not recorded in any poultry flock. Of 309 chicken sera tested, 11 (3.6%) from five farms across both regions tested positive for influenza A antibodies. In contrast, 16/54 (30%) duck sera from three farms in the Wairarapa were positive. Avian influenza H5 and H7 subtype-specific antibodies were excluded in ELISApositive sera using HI testing, and influenza A virus was not detected using RRT-PCR. CONCLUSIONS: The study confirmed that small backyard poultry flocks located near waterfowl habitats were exposed to non-notifiable low-pathogenic AI viruses. Findings indicate a number of potential risk pathways for the transmission of AI viruses between wild birds and non-commercial poultry, and hence the need for continued surveillance for AI in backyard flocks and wild birds in New Zealand.

Activity induced changes in the distribution of Shanks at hippocampal synapses

Dendritic spines contain a family of abundant scaffolding proteins known as Shanks, but little is known about how their distributions might change during synaptic activity. Here, pre-embedding immunogold electron microscopy is used to localize Shanks in synapses from cultured hippocampal neurons. We find that Shanks are preferentially located at postsynaptic densities (PSDs) as well as in a filamentous network near the PSD, extending up to 120 nm from the postsynaptic membrane. Application of sub-type specific antibodies shows that Shank2 is typically concentrated at and near PSDs while Shank1 is, in addition, distributed throughout the spine head. Depolarization with high K+ for 2 min causes transient, reversible translocation of Shanks towards the PSD that is dependent on extracellular Ca2+. The amount of activity-induced redistribution and subsequent recovery is pronounced for Shank1 but less so for Shank2. Thus, Shank1 appears to be a dynamic element within the spine, whose translocation could be involved in activity-induced, transient structural changes, while Shank2 appears to be a more stable element positioned at the interface of the PSD with the spine cytoplasm.

Dynamics of Specific Antibody Responses Induced in Mallards After Infection by or Immunization with Low Pathogenicity Avian Influenza Viruses

Natural infections with different subtypes of low pathogenicity avian influenza viruses (LPAIVs) are very common in wild duck populations. Recent outbreaks of high pathogenicity avian influenza virus (HPAIV) H5N1 in Eurasian and African countries stimulated monitoring activities in aquatic wild bird populations. Surveillance mainly focused on virus detection. Only a few serologic investigations have been conducted so far, although such data may retrospectively elucidate epidemiologic patterns of different AIV subtypes in the populations under study. To better understand the immunologic and serologic reactions of mallards after infection with LPAIV, we investigated the AIV type- and subtype-specific antibody dynamics in mallards after different LPAIV infections by hemagglutination inhibition, competitive enzyme-linked immunosorbent assay, and western blot analysis, as appropriate. Four groups of mallard ducks were used: 1) naturally infected birds, 2) birds that were experimentally infected with LPAIV, 3) birds that were immunized with inactivated virus preparations, and 4) negative control birds. Ducks were monitored for up to 15 mo, and serum samples were investigated every 1-4 wk. It could be shown that infection with LPAIV in mallards can be traced serologically over prolonged periods of time.

Plasma Exchange for Heparin-Induced Thrombocytopenia: Is There Enough Evidence?

Plasma Exchange for Heparin-Induced Thrombocytopenia: Is There Enough Evidence?

Abstract 5784: Altered Receptor Distribution and Expression Causing Vascular Myocytes Insensitivity to α 1 Adrenergic Activation

Alpha 1D -adrenergic receptor ( α 1D -AR) plays a critical role in controlling peripheral vascular resistance and blood pressure. However, unlike α 1A - and α 1B -AR subtypes that primarily locate at the plasma membrane and function efficiently, α 1D -ARs are accumulated within intracellular compartments and poorly functioned in vitro. Using fluo4, BODIPY prazosin and subtype-specific antibodies to evaluate the native α 1 AR regulated Ca 2+ signaling and their subcellular distribution in rat aortic and cardiac myocytes, we tested the hypothesis that α 1D -ARs are altered during cell isolation or/and culturing procedures, causing the responding discordance between tissue and cells. In aortic rings and freshly isolated myocytes α 1 AR agonist phenylephrine (PE, 10 −5 M) induced vasoconstriction, and intracellular Ca 2+ increase and cell shortening, which could be abolished by a specific antagonist of α 1D -AR BMY7378 (10 −6 M), respectively. The cellular response to PE, however, was decreased to 55.2±4.9% in 1-day-and 0% in ≥2-day-cultured aortic myocytes (n= 40, and 238), but remained in all the cultured neonatal rat cardiomyocytes (n= 267). Similarly as the α 1A -ARs in cultured cardiomyocytes, α 1D -ARs distributed both intracellularly and at the cell membrane in freshly dispersed aortic myocytes, but mostly redistributed intracellularly in cultured aortic myocytes. Treating the culture medium with 2% charcoal/dextran (C/D) for 48 h caused restorations of the cell surface α 1D -AR distribution in 34.67±4.1% (n=84) and partial Ca 2+ signaling response to PE in 29.5±0.2% (n=81, p<0.05 vs. no C/D) cultured cells, suggesting an essential requirement for membrane expressed α 1 ARs for their efficient intracellular coupling. Additionally, the α 1D -AR expression in the cultured aortic myocytes was reduced relative to the tissue. These results indicate that α 1D -ARs can primarily express at the cell membrane in native system but occur internalized into the cytoplasm and declined in expression in culture condition, resulting in the intracellular uncoupling to α 1 adrenergic activation in vitro. Charcoal/dextran in culture medium partially prevents this receptor subtype redistribution.