Heterologous Expression in Remodeled C. elegans: A Platform for Monoaminergic Agonist Identification and Anthelmintic Screening.

Monoamines, such as 5-HT and tyramine (TA), paralyze both free-living and parasitic nematodes when applied exogenously and serotonergic agonists have been used to clear Haemonchus contortus infections in vivo. Since nematode cell lines are not available and animal screening options are limited, we have developed a screening platform to identify monoamine receptor agonists. Key receptors were expressed heterologously in chimeric, genetically-engineered Caenorhabditis elegans, at sites likely to yield robust phenotypes upon agonist stimulation. This approach potentially preserves the unique pharmacologies of the receptors, while including nematode-specific accessory proteins and the nematode cuticle. Importantly, the sensitivity of monoamine-dependent paralysis could be increased dramatically by hypotonic incubation or the use of bus mutants with increased cuticular permeabilities. We have demonstrated that the monoamine-dependent inhibition of key interneurons, cholinergic motor neurons or body wall muscle inhibited locomotion and caused paralysis. Specifically, 5-HT paralyzed C. elegans 5-HT receptor null animals expressing either nematode, insect or human orthologues of a key Gαo-coupled 5-HT1-like receptor in the cholinergic motor neurons. Importantly, 8-OH-DPAT and PAPP, 5-HT receptor agonists, differentially paralyzed the transgenic animals, with 8-OH-DPAT paralyzing mutant animals expressing the human receptor at concentrations well below those affecting its C. elegans or insect orthologues. Similarly, 5-HT and TA paralyzed C. elegans 5-HT or TA receptor null animals, respectively, expressing either C. elegans or H. contortus 5-HT or TA-gated Cl- channels in either C. elegans cholinergic motor neurons or body wall muscles. Together, these data suggest that this heterologous, ectopic expression screening approach will be useful for the identification of agonists for key monoamine receptors from parasites and could have broad application for the identification of ligands for a host of potential anthelmintic targets

The 5-HT/SER-4-dependent inhibition of either the AIB interneurons or cholinergic motor neurons causes locomotory paralysis.

<p><b>A.</b> Confocal images of 5-HT <i>quint</i> expressing SER-4::GFP in the AIB interneurons (P<i>npr-9</i>)(A1) or cholinergic motor neurons (P<i>unc-17β</i>)(A2). GFP fluorescence (A2) or GFP fluorescence overlaid on DIC image (A1). The red stain in A2 is coelomocyte-specific RFP screening marker. <b>B.</b> Paralysis of wild type, mutant and transgenic <i>C</i>. <i>elegans</i> on hypotonic, non-NGM agar plates. Wild type, quadruple 5-HT receptor null animals expressing only SER-4 (SER-4 <i>quad</i>) or 5-HT <i>quint</i> expressing the <i>C</i>. <i>elegans</i> 5-HT₁-like receptor, SER-4, in either the cholinergic motor neurons (P<i>unc-17β</i>) or the two AIB interneurons (P<i>npr-9</i>) were examined for 5-HT (1 mM)-dependent paralysis as outlined in Methods. Data are presented as mean ± SE (n = 3).</p

PAPP paralyzes <i>C</i>. <i>elegans</i> via SER-4 and DOP-3.

<p><b>A-C.</b> Paralysis of wild type, mutant and transgenic <i>C</i>. <i>elegans</i> on hypotonic non-NGM agar plates. <b>A.</b> PAPP (0.5 mM)-dependent paralysis of wild-type, 5-HT <i>quint</i> and 5-HT <i>quint</i> animals expressing SER-4 in the cholinergic motor neurons (P<i>unc-17β</i>). Data are presented as mean ± SE (n = 3). <b>B.</b> Dose-response curves for PAPP-dependent paralysis at 15 min exposure for wild type, 5-HT <i>quint</i> and 5-HT <i>quint</i> animals expressing SER-4 in the cholinergic motor neurons (P<i>unc-17β</i>). <b>C.</b> PAPP (0.5 mM)-dependent paralysis of 5-HT <i>quint</i> and 5-HT <i>quint</i> animals expressing P<i>dop-3</i>::<i>dop-3</i> RNAi. Data are presented as mean ± SE (n = 3). ‘*’ p≤0.001, significantly different from 5-HT <i>quint</i> animals assayed under identical conditions.</p

5-HT and 5-HT receptor agonists selectively paralyze <i>C</i>. <i>elegans</i> 5-HT receptor mutant animals expressing nematode, insect or human 5-HT₁-like receptors in the cholinergic motor neurons.

<p><b>A-C.</b> Paralysis of wild type, mutant and transgenic <i>C</i>. <i>elegans</i> on hypotonic, non-NGM agar plates. <b>A.</b> 5-HT (1 mM)-dependent paralysis of 5-HT <i>quint</i> animals expressing either <i>C</i>. <i>elegans</i> 5-HT₁-like (SER-4), <i>Drosophila</i> 5-HT₁-like, or human 5-HT_1A receptor in cholinergic motor neurons (P<i>unc-17β</i>). Data are presented as mean ± SE (n = 3). <b>B.</b> 8-OH-DPAT (2 mM)-dependent paralysis of 5-HT <i>quint</i> animals expressing either <i>C</i>. <i>elegans</i> 5-HT₁-like (SER-4), <i>Drosophila</i> 5-HT₁-like, or human 5-HT_1A receptor in cholinergic motor neurons (P<i>unc-17β</i>). Data are presented as mean ± SE (n = 3). <b>C.</b> Sumatriptan (1 mM)-dependent paralysis of wild type, 5-HT <i>quint</i> animals expressing either <i>C</i>. <i>elegans</i> 5-HT₁-like (SER-4), <i>Drosophila</i> 5-HT₁-like, or human 5-HT_1A receptor in cholinergic motor neurons (P<i>unc-17β</i>). Data are presented as mean ± SE (n = 3).</p

<i>C</i>. <i>elegans</i> mutants with increased cuticular permeability are hypersensitive to 5-HT-dependent paralysis.

<p><b>A-B.</b> Paralysis of wild type and mutant <i>C</i>. <i>elegans</i> on NGM agar plates. <b>A.</b> Wild type animals examined for 5-HT-dependent paralysis as outlined in Methods. Data are presented as mean ± SE (n = 3). <b>B.</b> Dose-response curves for 5-HT-dependent paralysis on NGM plates at 10 min exposure for wild type and 5-HT <i>quint</i> animals. <b>C-D.</b> Paralysis of wild type and mutant <i>C</i>. <i>elegans</i> on non-NGM agar (hypotonic) plates. <b>C.</b> Wild type animals were examined for 5-HT-dependent paralysis as outlined in Methods. Data are presented as mean ± SE (n = 3). <b>D.</b> Dose-response curves for 5-HT-dependent paralysis in hypotonic conditions at 15 min exposure for wild type and 5-HT <i>quint</i> animals. <b>E-F.</b> 5-HT-dependent paralysis of wild type and mutant <i>C</i>. <i>elegans</i> on NGM agar plates. <b>E.</b> 5-HT (0.25 mM)-dependent paralysis of wild-type, <i>bus-8</i> (<i>e2968</i>), <i>bus-16</i> (<i>e2802</i>) and <i>bus-17</i> (<i>e2800</i>) mutants. Data are presented as mean ± SE (n = 3). <b>F.</b> Dose-response curves for 5-HT-dependent paralysis at 10 min exposure for wild type and <i>bus</i> mutants.</p

Platelet-T cell aggregates in lung cancer patients: Implications for thrombosis.

Platelet-leukocyte aggregates (PLAs) are associated with increased thrombosis risk. The influence of PLA formation is especially important for cancer patients, since thrombosis accounts for approximately 10% of cancer-associated deaths. Our objective was to characterize and quantify PLAs in whole blood samples from lung cancer patients compared to healthy volunteers with the intent to analyze PLA formation in the context of lung cancer-associated thrombosis. Consenting lung cancer patients (57) and healthy volunteers (56) were enrolled at the Dana Cancer Center at the University of Toledo Health Science Campus. Peripheral blood samples were analyzed by flow cytometry. Patient medical history was reviewed through electronic medical records. Most importantly, we found lung cancer patients to have higher percentages of platelet-T cell aggregates (PTCAs) than healthy volunteers among both CD4+ T lymphocyte and CD8+ T lymphocyte populations. Our findings demonstrate that characterization of PTCAs may have clinical utility in differentiating lung cancer patients from healthy volunteers and stratifying lung cancer patients by history of thrombosis

Targeted disruption of Cd40 in a genetically hypertensive rat model attenuates renal fibrosis and proteinuria, independent of blood pressure

High blood pressure is a common cause of chronic kidney disease. Because CD40, a member of the tumor necrosis factor receptor family, has been linked to the progression of kidney disease in ischemic nephropathy, we studied the role of Cd40 in the development of hypertensive renal disease. The Cd40 gene was mutated in the Dahl S genetically hypertensive rat with renal disease by targeted-gene disruption using zinc-finger nuclease technology. These rats were then given low (0.3%) and high (2%) salt diets and compared. The resultant Cd40 mutants had significantly reduced levels of both urinary protein excretion (41.8 ± 3.1 mg/24 h vs. 103.7 ± 4.3 mg/24 h) and plasma creatinine (0.36 ± 0.05 mg/dl vs. 1.15 ± 0.19 mg/dl), with significantly higher creatinine clearance compared with the control S rats (3.04 ± 0.48 ml/min vs. 0.93 ± 0.15 ml/min), indicating renoprotection was conferred by mutation of the Cd40 locus. Furthermore, the Cd40 mutants had a significant attenuation in renal fibrosis, which persisted on the high salt diet. However, there was no difference in systolic blood pressure between the control and Cd40 mutant rats. Thus, these data serve as the first evidence for a direct link between Cd40 and hypertensive nephropathy. Hence, renal fibrosis is one of the underlying mechanisms by which Cd40 plays a crucial role in the development of hypertensive renal disease