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Hookworm infection is associated with anaemia and malnutrition in many resource-limited countries. Ancylostoma hookworms have previously been shown to modulate host cellular immune responses through multiple mechanisms, including reduced mitogen-mediated lymphocyte proliferation, impaired antigen presentation/processing, and relative reductions in CD4+ T cells in the spleen and mesenteric lymph nodes. Syrian hamsters were depleted of CD4+ for up to 9 days following intraperitoneal injection (200 μg) of a murine anti-mouse CD4 monoclonal IgG (clone GK1·5). CD4+ T-cell-depleted hamsters infected with the hookworm Ancylostoma ceylanicum exhibited a threefold higher mean intestinal worm burden and more severe anaemia than animals that received isotype control IgG. In addition, depletion of CD4+ T cells was associated with impaired cellular and humoral (serum and mucosal) immune responses to hookworm antigens. These data demonstrate an effector role for CD4+ T cells in hookworm immunity and disease pathogenesis. Ultimately, these studies may yield important insights into the relationship between intestinal nematode infections and diseases that are associated with CD4+ T-cell depletion, including HIV. [ABSTRACT FROM AUTHOR]

Abstract Background The Nef protein of HIV facilitates virus replication and disease progression in infected patients. This role as pathogenesis factor depends on several genetically separable Nef functions that are mediated by interactions of highly conserved protein-protein interaction motifs with different host cell proteins. By studying the functionality of a series of nef alleles from clinical isolates, we identified a dysfunctional HIV group O Nef in which a highly conserved valine-glycine-phenylalanine (VGF) region, which links a preceding acidic cluster with the following proline-rich motif into an amphipathic surface was deleted. In this study, we aimed to study the functional importance of this VGF region. Results The dysfunctional HIV group O8 nef allele was restored to the consensus sequence, and mutants of canonical (NL4.3, NA-7, SF2) and non-canonical (B2 and C1422) HIV-1 group M nef alleles were generated in which the amino acids of the VGF region were changed into alanines (VGF→AAA) and tested for their capacity to interfere with surface receptor trafficking, signal transduction and enhancement of viral replication and infectivity. We found the VGF motif, and each individual amino acid of this motif, to be critical for downregulation of MHC-I and CXCR4. Moreover, Nef’s association with the cellular p21-activated kinase 2 (PAK2), the resulting deregulation of cofilin and inhibition of host cell actin remodeling, and targeting of Lck kinase to the trans-golgi-network (TGN) were affected as well. Of particular interest, VGF integrity was essential for Nef-mediated enhancement of HIV virion infectivity and HIV replication in peripheral blood lymphocytes. For targeting of Lck kinase to the TGN and viral infectivity, especially the phenylalanine of the triplet was essential. At the molecular level, the VGF motif was required for the physical interaction of the adjacent proline-rich motif with Hck. Conclusion Based on these findings, we propose that this highly conserved three amino acid VGF motif together with the acidic cluster and the proline-rich motif form a previously unrecognized amphipathic surface on Nef. This surface appears to be essential for the majority of Nef functions and thus represents a prime target for the pharmacological inhibition of Nef.

Interferon (IFN) inhibits HIV replication by inducing antiviral effectors. To comprehensively identify IFN-induced HIV restriction factors, we assembled a CRISPR sgRNA library of Interferon Stimulated Genes (ISGs) into a modified lentiviral vector that allows for packaging of sgRNA-encoding genomes in trans into budding HIV-1 particles. We observed that knockout of Zinc Antiviral Protein (ZAP) improved the performance of the screen due to ZAP-mediated inhibition of the vector. A small panel of IFN-induced HIV restriction factors, including MxB, IFITM1, Tetherin/BST2 and TRIM5alpha together explain the inhibitory effects of IFN on the CXCR4-tropic HIV-1 strain, HIV-1 LAI , in THP-1 cells. A second screen with a CCR5-tropic primary strain, HIV-1 Q23.BG505 , described an overlapping, but non-identical, panel of restriction factors. Further, this screen also identifies HIV dependency factors. The ability of IFN-induced restriction factors to inhibit HIV strains to replicate in human cells suggests that these human restriction factors are incompletely antagonized., Editorial Note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter)., Competing Interests: MO, LH, JV, FR, DH, RB, JD, JO, ME No competing interests declared, (© 2018, OhAinle et al.)

Several pattern-recognition receptors sense HIV-1 replication products and induce type I interferon (IFN-I) production under specific experimental conditions. However, it is thought that viral sensing and IFN induction are virtually absent in the main target cells of HIV-1 in vivo. Here, we show that activated CD4 + T cells sense HIV-1 infection through the cytosolic DNA sensor cGAS and mount a bioactive IFN-I response. Efficient induction of IFN-I by HIV-1 infection requires proviral integration and is regulated by newly expressed viral accessory proteins: Vpr potentiates, while Vpu suppresses cGAS-dependent IFN-I induction. Furthermore, Vpr also amplifies innate sensing of HIV-1 infection in Vpx-treated dendritic cells. Our results identify cGAS as mediator of an IFN-I response to HIV-1 infection in CD4 + T cells and demonstrate that this response is modulated by the viral accessory proteins Vpr and Vpu. Thus, viral innate immune evasion is incomplete in the main target cells of HIV-1., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

The majority of T cells encountered by HIV-1 are non-activated and do not readily allow productive infection. HIV-1 Vpr is highly abundant in progeny virions, and induces signalling and HIV-1 LTR transcription. We hence hypothesized that Vpr might be a determinant of non-activated T-cell infection. Virion-delivered Vpr activated nuclear factor of activated T cells (NFAT) through Ca(2+) influx and interference with the NFAT export kinase GSK3β. This leads to NFAT translocation and accumulation within the nucleus and was required for productive infection of unstimulated primary CD4(+) T cells. A mutagenesis approach revealed correlation of Vpr-mediated NFAT activation with its ability to enhance LTR transcription and mediate cell cycle arrest. Upon NFAT inhibition, Vpr did not augment resting T-cell infection, and showed reduced G2/M arrest and LTR transactivation. Altogether, Vpr renders unstimulated T cells more permissive for productive HIV-1 infection and stimulates activation of productively infected as well as virus-exposed T cells. Therefore, it could be involved in the establishment and reactivation of HIV-1 from viral reservoirs and might have an impact on the levels of immune activation, which are determinants of HIV-1 pathogenesis., (© 2016 The Authors.)

The function of dendritic cells (DCs) in the immune system is based on their ability to sense and present foreign antigens. Powerful tools to research DC function and to apply in cell-based immunotherapy are either silencing or overexpression of genes achieved by lentiviral transduction. To date, efficient lentiviral transduction of DCs or their monocyte derived counterparts (MDDCs) required high multiplicity of infection (MOI) or the exposure to the HIV-2/SIV protein Vpx to degrade viral restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1). Here we present a Vpx-independent method for efficient (>95%) transduction of MDDCs at lower MOI. The protocol can be used both for ectopic gene expression and knock-down. Introducing shRNA targeting viral entry receptor CD4 and restriction factor SAMHD1 into MDDCs resulted in down-regulation of targeted proteins and, consequently, expected impact on HIV infection. This protocol for MDDCs transduction is robust and free of the potential risk arising from the use of Vpx which creates a virus infection-prone environment, potentially dangerous in clinical setting.

Episomal HIV-1 two-long terminal repeat (2-LTR) circles are considered markers for ongoing viral replication. Two sample processing procedures were compared to accurately quantify 2-LTR in patients by using droplet digital PCR (ddPCR). Here, we show that plasmid isolation with a spiked non-HIV plasmid for normalization enables more accurate 2-LTR quantification than genomic DNA isolation., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Down-modulation of the CD4 receptor is one of the hallmarks of HIV-1 infection and it is believed to confer a selective replicative advantage to the virus in vivo. This process is mainly mediated by three viral proteins: Env, Vpu and Nef. To date, the mechanisms that lead to CD4 depletion from the surface of infected cells during HIV-1 infection are still only partially characterized. In this study, we sought to identify and characterize cellular host factors in HIV-1-induced CD4 down-modulation., Results: To identify host factors involved in CD4 down-regulation, we used a whole genome-targeting shRNA lentiviral library in HeLa CD4+ cells expressing Nef as an inducer of CD4 down-modulation. We identified 55 genes, mainly encoding for proteins involved in various steps of clathrin-mediated endocytosis. For confirmation and further selection of the hits we performed several rounds of validation, using individual shRNA lentiviral vectors with a different target sequence for gene knock-down in HIV-1-infected T cells. By this stringent validation set-up, we could demonstrate that the knock-down of DNM3 (dynamin 3), SNX22 (sorting nexin 22), ATP6AP1 (ATPase, H+ Transporting, Lysosomal Accessory Protein 1), HRBL (HIV-Rev binding protein Like), IDH3G (Isocitrate dehydrogenase), HSP90B1 (Heat shock protein 90 kDa beta member 1) and EPS15 (Epidermal Growth Factor Receptor Pathway Substrate 15) significantly increases CD4 levels in HIV-infected SupT1 T cells compared to the non-targeting shRNA control. Moreover, EPS15, DNM3, IDH3G and ATP6AP1 knock-down significantly decreases HIV-1 replication in T cells., Conclusions: We identified seven genes as cellular co-factors for HIV-1-mediated CD4 down-regulation in T cells. The knock-down of four out of seven of these genes also significantly reduces HIV-1 replication in T cells. Next to a role in HIV-mediated CD4 down-regulation, these genes might however affect HIV-1 replication in another way. Our findings give insights in the HIV-1-mediated CD4 down-regulation at the level of the plasma membrane and early endosomes and identify four possible new HIV-1 replication co-factors.

Introduction: In HIV-infected patients on combination antiretroviral therapy (cART), the detection of episomal HIV 2-LTR circles is a potential marker for ongoing viral replication. Quantification of 2-LTR circles is based on quantitative PCR or more recently on digital PCR assessment, but is hampered due to its low abundance. Sample pre-PCR processing is a critical step for 2-LTR circles quantification, which has not yet been sufficiently evaluated in patient derived samples., Materials and Methods: We compared two sample processing procedures to more accurately quantify 2-LTR circles using droplet digital PCR (ddPCR). Episomal HIV 2-LTR circles were either isolated by genomic DNA isolation or by a modified plasmid DNA isolation, to separate the small episomal circular DNA from chromosomal DNA. This was performed in a dilution series of HIV-infected cells and HIV-1 infected patient derived samples (n=59). Samples for the plasmid DNA isolation method were spiked with an internal control plasmid., Results: Genomic DNA isolation enables robust 2-LTR circles quantification. However, in the lower ranges of detection, PCR inhibition caused by high genomic DNA load substantially limits the amount of sample input and this impacts sensitivity and accuracy. Moreover, total genomic DNA isolation resulted in a lower recovery of 2-LTR templates per isolate, further reducing its sensitivity. The modified plasmid DNA isolation with a spiked reference for normalization was more accurate in these low ranges compared to genomic DNA isolation. A linear correlation of both methods was observed in the dilution series (R2=0.974) and in the patient derived samples with 2-LTR numbers above 10 copies per million peripheral blood mononuclear cells (PBMCs), (R2=0.671). Furthermore, Bland-Altman analysis revealed an average agreement between the methods within the 27 samples in which 2-LTR circles were detectable with both methods (bias: 0.3875±1.2657 log10)., Conclusions: 2-LTR circles quantification in HIV-infected patients proved to be more accurate with a modified plasmid DNA isolation procedure compared to total genomic DNA isolation. This method enables the processing of more blood cells, thus enhancing quantification accuracy and sensitivity. An improved quantification of 2-LTR circles will contribute to the better understanding of ongoing replication in the HIV reservoir of patients on cART.

Adenosine A2A antagonists are believed to have therapeutic potential in the treatment of Parkinson's disease (PD). We have characterized the dual adenosine A2A/A1 receptor antagonist JNJ-40255293 (2-amino-8-[2-(4-morpholinyl)ethoxy]-4-phenyl-5H-indeno[1,2-d]pyrimidin-5-one). JNJ-40255293 was a high-affinity (7.5 nM) antagonist at the human A2A receptor with 7-fold in vitro selectivity versus the human A1 receptor. A similar A2A:A1 selectivity was seen in vivo (ED50's of 0.21 and 2.1 mg/kg p.o. for occupancy of rat brain A2A and A1 receptors, respectively). The plasma EC50 for occupancy of rat brain A2A receptors was 13 ng/mL. In sleep-wake encephalographic (EEG) studies, JNJ-40255293 dose-dependently enhanced a consolidated waking associated with a subsequent delayed compensatory sleep (minimum effective dose: 0.63 mg/kg p.o.). As measured by microdialysis, JNJ-40255293 did not affect dopamine and noradrenaline release in the prefrontal cortex and the striatum. However, it was able to reverse effects (catalepsy, hypolocomotion, and conditioned avoidance impairment in rats; hypolocomotion in mice) produced by the dopamine D2 antagonist haloperidol. The compound also potentiated the agitation induced by the dopamine agonist apomorphine. JNJ-40255293 also reversed hypolocomotion produced by the dopamine-depleting agent reserpine and potentiated the effects of l-dihydroxyphenylalanine (L-DOPA) in rats with unilateral 6-hydroxydopamine-induced lesions of the nigro-striatal pathway, an animal model of Parkinson's disease. Extrapolating from the rat receptor occupancy dose-response curve, the occupancy required to produce these various effects in rats was generally in the range of 60-90%. The findings support the continued research and development of A2A antagonists as potential treatments for PD.

The central memory T cell (TCM) model forms a unique HIV-1 latency model based on primary cells that closely resemble in vivo TCM. The virus employed in this model is based on an engineered vector incapable of replication after initial infection. We show that despite this strategy, replication competent viral particles are released into the culture medium due to recombination between overlapping sequences of the env deleted HIV genome that is co-transfected with intact env. This finding emphasizes the need for careful data analysis and interpretation if similar constructs are employed and urges for additional caution during laboratory work.

Chronic lymphocytic leukaemia (CLL) is a disease with a highly variable prognosis. The clinical course can however be predicted thanks to prognostic markers. Poor prognosis is associated with expression of a B cell receptor (BCR) from unmutated immunoglobulin variable heavy-chain genes (IGHV) and expression of zeta-associated protein of 70 kDa (ZAP70). The reason why ZAP70 expression is associated with poor prognosis and whether the protein has a direct pathogenic function is at present unknown. By transfer of ZAP70 to CLL cells, we show here that expression of ZAP70 in CLL cells leads to increased expression of the nuclear factor (NF)-κB target genes interleukin-1β (IL1B), IL6 and IL8 upon BCR triggering. This could be blocked by inhibition of NF-κB signalling through inhibition of IκB kinases (IKK). Transcriptome analysis identified a NF-κB RELA signature imposed by ZAP70 expression in BCR-stimulated CLL cells. We conclude that ZAP70 acts directly as an amplifier of NF-κB signalling in CLL cells which could be an underlying mechanism for its association with poor prognosis and which may represent a therapeutic target., (© 2013 John Wiley & Sons Ltd.)

Chronic lymphocytic leukemia (CLL) is a disease with variable clinical outcome. Several prognostic factors such as the immunoglobulin heavy chain variable genes (IGHV) mutation status are linked to the B-cell receptor (BCR) complex, supporting a role for triggering the BCR in vivo in the pathogenesis. The miRNA profile upon stimulation and correlation with IGHV mutation status is however unknown. To evaluate the transcriptional response of peripheral blood CLL cells upon BCR stimulation in vitro, miRNA and mRNA expression was measured using hybridization arrays and qPCR. We found both IGHV mutated and unmutated CLL cells to respond with increased expression of MYC and other genes associated with BCR activation, and a phenotype of cell cycle progression. Genome-wide expression studies showed hsa-miR-132-3p/hsa-miR-212 miRNA cluster induction associated with a set of downregulated genes, enriched for genes modulated by BCR activation and amplified by Myc. We conclude that BCR triggering of CLL cells induces a transcriptional response of genes associated with BCR activation, enhanced cell cycle entry and progression and suggest that part of the transcriptional profiles linked to IGHV mutation status observed in isolated peripheral blood are not cell intrinsic but rather secondary to in vivo BCR stimulation.

Hookworm disease, characterized by severe anemia and cognitive and growth delays, currently affects an estimated 740 million people worldwide. Despite the prevalence of this parasitic disease, few effective drug therapies are in use today, and the heavy reliance upon benzimidazoles highlights the need for the development of novel chemotherapies. Recent work with the trematode parasite Schistosoma mansoni has identified oxadiazole 2-oxides as effective antischistosomal compounds that function by targeting and inhibiting the antioxidant enzyme, thioredoxin glutathione reductase. In this study, a related enzyme, glutathione reductase, from the human hookworm Ancylostoma ceylanicum was identified and characterized, and its in vitro activity in the presence of the oxadiazole 2-oxides was analyzed. Ex vivo worm killing assays were also conducted to establish the relationship between a given compound's effect upon worm survival and inhibition of recombinant glutathione reductase (rAceGR). Finally, the in vivo anthelminthic efficacy of furoxan (Fx) was assessed in the hamster model of hookworm infection. The predicted amino acid sequence of AceGR contained a prototypical glutathione reductase active site sequence, but no thioredoxin reductase consensus sequences, suggesting that the glutathione and thioredoxin pathways of A. ceylanicum are distinct. Although ten of the forty-two oxadiazole 2-oxides tested inhibited rAceGR activity by at least fifty percent, and fifteen compounds were toxic to parasites ex vivo, little overlap existed between these two results. We therefore suggest that AceGR is not the primary target of the oxadiazole 2-oxides in effecting parasite death. Lastly, oral treatment of A. ceylanicuminfected hamsters with furoxan resulted in significantly improved weight gains and reduced intestinal worm burdens compared to vehicle treated controls, supporting continued development of this molecule as a novel anthelminthic.

All marketed antipsychotics act by blocking dopamine D(2) receptors. Fast dissociation from D(2) receptors may be one of the elements contributing to the lower incidence of extrapyramidal symptoms (EPS) exhibited by newer antipsychotics. Therefore, we screened for specific D(2) receptor blockers with a fast rate of dissociation. Radioligand binding experiments identified N-[1-(3,4-difluorobenzyl)piperidin-4-yl]-6-(trifluoromethyl)pyridazin-3-amine (JNJ-37822681) as a fast-dissociating D(2) ligand. Its D(2) receptor specificity was high compared with atypical antipsychotics, with little activity at receptors associated with unwanted effects [α(1), α(2), H(1), muscarinic, and 5-hydroxytryptamine (5-HT) type 2C] and for receptors that may interfere with the effects of D(2) antagonism (D(1), D(3), and 5-HT(2A)). JNJ-37822681 occupied D(2) receptors in rat brain at relatively low doses (ED(50) 0.39 mg/kg) and was effective in animal models of psychosis (e.g., inhibition of apomorphine-induced stereotypy or D-amphetamine/phencyclidine-induced hyperlocomotion). Prolactin levels increased from an ED(50) (0.17 mg/kg, peripheral D(2) receptors) close to the ED(50) required for apomorphine antagonism (0.19 mg/kg, central D(2) receptors), suggesting excellent brain disposition and minimal prolactin release at therapeutic doses. JNJ-37822681 induced catalepsy and inhibited avoidance behavior, but with a specificity margin relative to apomorphine antagonism that was larger than that obtained for haloperidol and similar to that obtained for olanzapine. This larger specificity margin (compared with haloperidol) may reflect lower EPS liability and less behavioral suppression after JNJ-37822681. JNJ-37822681 is a novel, potent, specific, centrally active, fast-dissociating D(2) antagonist with optimal brain disposition, and it is the first compound that allows the evaluation of the potential value of fast D(2) antagonism for the treatment of schizophrenia and bipolar disorder.

Quantification of retroviruses in cell culture supernatants and other biological preparations is required in a diverse spectrum of laboratories and applications. Methods based on antigen detection, such as p24 for HIV, or on genome detection are virus specific and sometimes suffer from a limited dynamic range of detection. In contrast, measurement of reverse transcriptase (RT) activity is a generic method which can be adapted for higher sensitivity using real-time PCR quantification (qPCR-based product-enhanced RT (PERT) assay). We present an evaluation of a modified SYBR Green I-based PERT assay (SG-PERT), using commercially available reagents such as MS2 RNA and ready-to-use qPCR mixes. This assay has a dynamic range of 7 logs, a sensitivity of 10 nU HIV-1 RT and outperforms p24 ELISA for HIV titer determination by lower inter-run variation, lower cost and higher linear range. The SG-PERT values correlate with transducing and infectious units in HIV-based viral vector and replication-competent HIV-1 preparations respectively. This assay can furthermore quantify Moloney Murine Leukemia Virus-derived vectors and can be performed on different instruments, such as Roche Lightcycler® 480 and Applied Biosystems ABI 7300. We consider this test to be an accurate, fast and relatively cheap method for retroviral quantification that is easily implemented for use in routine and research laboratories.

The Nef protein is an essential factor for lentiviral pathogenesis in humans and other simians. Despite a multitude of functions attributed to this protein, the exact role of Nef in disease progression remains unclear. One of its most intriguing functions is the ability of Nef to enhance the infectivity of viral particles. In this review we will discuss current insights in the mechanism of this well-known, yet poorly understood Nef effect. We will elaborate on effects of Nef, on both virion biogenesis and the early stage of the cellular infection, that might be involved in infectivity enhancement. In addition, we provide an overview of different HIV-1 Nef domains important for optimal infectivity and briefly discuss some possible sources of the frequent discrepancies in the field. Hereby we aim to contribute to a better understanding of this highly conserved and therapeutically attractive Nef function.

Complex intra-articular fractures of the proximal tibia are difficult to treat, especially in the elderly osteoporotic patient. Pre-existing osteoarthritis, cartilage damage during trauma, suboptimal reduction and fixation due to poor bone stock and/or secondary displacement frequently lead to poor outcome. After osteosynthesis rehabilitation is cumbersome as patients have been non-weight bearing for long periods of time and secondary total knee arthroplasty can be challenging. For these reasons, we investigated the possibility to perform a total knee arthroplasty with or without adjuvant osteosynthesis as a primary treatment in elderly and/or osteoarthritic patients with complex tibial plateau fractures. Between 2002 and 2009, 12 patients (mean age: 73 years (58-81)) with an AO-41 fracture type B1 (1), B3 (8) and C3 (3) were treated with a primary total knee arthroplasty within three weeks from their trauma. Most patients (7/12) were allowed early full-weight bearing. One patient died due to an unrelated cause; the remaining eleven were reviewed at a mean follow-up period of 31 months (5 w-81 m). At final follow-up the median knee score was 78 (50-100) and the function score 58 (0-100): 7/11 patients had an excellent result, while 1/11 had a fair and 3/11 a poor result. Fair and poor results were mostly related to pre-existing poor general condition and/or concomitant disease. Most patients were satisfied and only minor short- and long-term complications were noted. There was no need for revision surgery. Our limited series of well-selected elderly and/or osteoarthritic patients with a complex tibial plateau fracture treated with primary total knee arthroplasty yielded encouraging results.

Parasitic organisms have evolved specialized strategies to evade immune defense mechanisms. We describe herein an ortholog of the cytokine, macrophage migration inhibitory factor (MIF), which is produced by the obligate intracellular parasite, Leishmania major. The Leishmania MIF protein, Lm1740MIF, shows significant structural homology with human MIF as revealed by a high-resolution x-ray crystal structure (1.03 A). Differences between the two proteins in the N-terminal tautomerization site are evident, and we provide evidence for the selective, species-specific inhibition of MIF by small-molecule antagonists that target this site. Lm1740MIF shows significant binding interaction with the MIF receptor, CD74 (K(d) = 2.9 x 10(-8) M). Like its mammalian counterpart, Lm1740MIF induces ERK1/2 MAP kinase activation in a CD74-dependent manner and inhibits the activation-induced apoptosis of macrophages. The ability of Lm1740MIF to inhibit apoptosis may facilitate the persistence of Leishmania within the macrophage and contribute to its evasion from immune destruction.

Clinical studies report a low incidence of intestinal side effects with transdermally administered fentanyl (TTS-fentanyl) in comparison with oral morphine. To support these clinical data, analgesic and intestinal effects of both opioids were compared in rats. After subcutaneous injection, analgesia in the tail withdrawal reaction test was obtained at a peak effect dose of 0.032 mg/kg with fentanyl and 8.0 mg/kg with morphine. This analgesic dose exceeded the ED50 for inhibition of castor oil-induced diarrhea only slightly (1.1 x) in the case of fentanyl (0.028 mg/kg) but markedly (36 x) in the case of morphine (0.22 mg/kg). To reverse completely the antidiarrheal effect of equivalent analgesic doses of the opioids (their ED50S for analgesia lasting 2 hours), much more naloxone was required in the case of morphine (5.4 mg/kg) than in the case of fentanyl (0.19 mg/kg). After oral administration, the difference between both opioids was less pronounced. Analgesia was obtained at 0.85 mg/kg with fentanyl and 32 mg/kg with morphine. This analgesic dose only slightly (1.7 x) exceeded the antidiarrheal dose in the case of fentanyl (0.49 mg/kg) but significantly (6.2 x) in the case of morphine (5.2 mg/ kg). To reverse completely the antidiarrheal effect of equivalent analgesic oral doses of the opioids (their ED50S for analgesia lasting 2 hours), more naloxone was required in the case of morphine (11 mg/kg) than in the case of fentanyl (2.0 mg/kg). Rapid penetration of fentanyl into the brain is thought to be responsible for small dissociation between the analgesic and intestinal effect of this lipophilic opioid. The present data provide preclinical evidence to support the relatively low incidence of intestinal side effects observed clinically with the use of TTS-fentanyl in comparison with orally administered morphine.

A systematic study of a large number of compounds from various pharmacological classes was performed to better define their interaction with the cholinergic nervous system. An 'in vivo' test procedure called 'physostigmine antagonism' in rats was used; it involved the administration of the test compounds, measurement of the pupil diameter and recording of the survival time after injection of a lethal dose of physostigmine. Known peripherally acting anticholinergics, such as isopropamide and methylscopolamine did not protect from physostigmine lethality at doses up to more than 150 times the mydriatic dose. Known centrally acting anticholinergics, such as dexetimide and benztropine, protected from lethality at doses equal to or slightly higher than the mydriatic dose. Penetration into the brain of a muscarinic blocking agent thus appeared to be a sufficient condition to significantly reduce the cholinergic overstimulation of the CNS that results from inhibition of acetylcholine hydrolysis. Drugs of other pharmacological classes that are known to have anticholinergic activity in addition to their more characteristic action, were also active in the physostigmine test. They include most of the tricyclic antidepressants, some antihistamines such as diphenhydramine and cyproheptadine, some ganglion blocking agents such as mecamylamine and the neuroleptic clozapine. Drugs with hypnotic or anticonvulsant properties, sedative neuroleptics and high doses of some members of other pharmacological classes protected from physostigmine-induced lethality by a mechanism not based on anticholinergic activity. The results further show that a number of pharmacological actions: dopamine, histamine H1 and serotonin S2 antagonism, MAO-inhibition, alpha-adrenergic blockade etc. are all insufficient to produce physostigmine antagonism.