Trends in the use of laboratory tests for the diagnosis of Clostridium difficile infection and association with incidence rates in Quebec, Canada, 2010-2014.

BACKGROUND: Several Clostridium difficile infection (CDI) surveillance programs do not specify laboratory strategies to use. We investigated the evolution in testing strategies used across Quebec, Canada, and its association with incidence rates. METHODS: Cross-sectional study of 95 hospitals by surveys conducted in 2010 and in 2013-2014. The association between testing strategies and institutional CDI incidence rates was analyzed via multivariate Poisson regressions. RESULTS: The most common assays in 2014 were toxin A/B enzyme immunoassays (EIAs) (61 institutions, 64%), glutamate dehydrogenase (GDH) EIAs (51 institutions, 53.7%), and nucleic acid amplification tests (NAATs) (34 institutions, 35.8%). The most frequent algorithm was a single-step NAAT (20 institutions, 21%). Between 2010 and 2014, 35 institutions (37%) modified their algorithm. Institutions detecting toxigenic C difficile instead of C difficile toxin increased from 14 to 37 (P < .001). Institutions detecting toxigenic C difficile had higher CDI rates (7.9 vs 6.6 per 10,000 patient days; P = .01). Institutions using single-step NAATs, GDH plus toxigenic cultures, and GDH plus cytotoxicity assays had higher CDI rates than those using an EIA-based algorithm (P < .05). CONCLUSIONS: Laboratory detection of CDI has changed since 2010. There is an association between diagnostic algorithms and CDI incidence. Mitigation strategies are warranted.

Loss-of-function mutations of Dynamin 2 promote T-ALL by enhancing IL-7 signalling.

Mutations in the DYNAMIN2 (DNM2) gene are frequently detected in human acute T-cell lymphoblastic leukemia (T-ALL), although the mechanisms linking these mutations to disease pathogenesis remain unknown. Using an ENU-based forward genetic screen for mice with erythroid phenotypes, we identified a heterozygous mouse line carrying a mutation in the GTPase domain of Dnm2 (Dnm2V265G) that induced a microcytic anemia. In vitro assays using the V265G mutant demonstrated loss of GTPase activity and impaired endocytosis that was comparable to other DNM2 mutants identified in human T-ALL. To determine the effects of DNM2 mutations in T-ALL, we bred the Dnm2V265G mice with the Lmo2 transgenic mouse model of T-ALL. Heterozygous Dnm2 mutants lacking the Lmo2 transgene displayed normal T-cell development, and did not develop T-ALL. In contrast, compound heterozygotes displayed an accelerated onset of T-ALL compared with mice carrying the Lmo2 oncogene alone. The leukemias from these mice exhibited a more immature immunophenotype and an expansion in leukemic stem cell numbers. Mechanistically, the Dnm2 mutation impaired clathrin-mediated endocytosis of the interleukin (IL)-7 receptor resulting in increased receptor density on the surface of leukemic stem cells. These findings suggest that DNM2 mutations cooperate with T-cell oncogenes by enhancing IL-7 signalling.

PUMA promotes apoptosis of hematopoietic progenitors driving leukemic progression in a mouse model of myelodysplasia.

Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis with resultant cytopenias. Increased apoptosis and aberrantly functioning progenitors are thought to contribute to this phenotype. As is the case for other malignancies, overcoming apoptosis is believed to be important in progression toward acute myeloid leukemia (AML). Using the NUP98-HOXD13 (NHD13) transgenic mouse model of MDS, we previously reported that overexpression of the anti-apoptotic protein BCL2, blocked apoptosis and improved cytopenias, paradoxically, delaying leukemic progression. To further understand this surprising result, we examined the role of p53 and its pro-apoptotic effectors, PUMA and NOXA in NHD13 mice. The absence of p53 or PUMA but not NOXA reduced apoptosis and expanded the numbers of MDS-repopulating cells. Despite a similar effect on apoptosis and cell numbers, the absence of p53 and PUMA had diametrically opposed effects on progression to AML: absence of p53 accelerated leukemic progression, while absence of PUMA significantly delayed progression. This may be explained in part by differences in cellular responses to DNA damage. The absence of p53 led to higher levels of γ-H2AX (indicative of persistent DNA lesions) while PUMA-deficient NHD13 progenitors resolved DNA lesions in a manner comparable to wild-type cells. These results suggest that targeting PUMA may improve the cytopenias of MDS without a detrimental effect on leukemic progression thus warranting further investigation.

Soluble CD40-ligand (sCD40L, sCD154) plays an immunosuppressive role via regulatory T cell expansion in HIV infection.

CD40/CD40-ligand (CD40L) signalling is a key stimulatory pathway which triggers the tryptophan (Trp) catabolizing enzyme IDO in dendritic cells and is immunosuppressive in cancer. We reported IDO-induced Trp catabolism results in a T helper type 17 (Th17)/regulatory T cell (Treg ) imbalance, and favours microbial translocation in HIV chronic infection. Here we assessed the link between sCD40L, Tregs and IDO activity in HIV-infected patients with different clinical outcomes. Plasmatic sCD40L and inflammatory cytokines were assessed in anti-retroviral therapy (ART)-naive, ART-successfully treated (ST), elite controllers (EC) and healthy subjects (HS). Plasma levels of Trp and its metabolite Kynurenine (Kyn) were measured by isotope dilution tandem mass spectrometry and sCD14 was assessed by enzyme-linked immunosorbent assay (ELISA). IDO-mRNA expression was quantified by reverse transcription-polymerase chain reaction (RT-PCR). The in-vitro functional assay of sCD40L on Treg induction and T cell activation were assessed on peripheral blood mononuclear cells (PBMCs) from HS. sCD40L levels in ART-naive subjects were significantly higher compared to ST and HS, whereas EC showed only a minor increase. In ART-naive alone, sCD40L was correlated with T cell activation, IDO-mRNA expression and CD4 T cell depletion but not with viral load. sCD40L was correlated positively with IDO enzymatic activity (Kyn/Trp ratio), Treg frequency, plasma sCD14 and inflammatory soluble factors in all HIV-infected patients. In-vitro functional sCD40L stimulation induced Treg expansion and favoured Treg differentiation by reducing central memory and increasing terminal effector Treg proportion. sCD40L also increased T cell activation measured by co-expression of CD38/human leucocyte antigen D-related (HLA-DR). These results indicate that elevated sCD40L induces immunosuppression in HIV infection by mediating IDO-induced Trp catabolism and Treg expansion.

Design and implementation of a randomized crossover study of valproic acid and antiretroviral therapy to reduce the HIV reservoir.

BACKGROUND: HIV reservoirs represent the major obstacles for eradication and are defined as a cell type that allows persistence of replication-competent HIV in patients on optimal long-term antiretroviral therapy (HAART). Several pilot clinical trials have been implemented to assess the value of experimental therapy to reduce reservoir size or eradicate HIV. In order to eradicate HIV, valproic acid was used as a new strategy to increase viral gene expression in the nucleus of infected cells with the expectation of generating a direct cell death or destruction by nearby cytotoxic cells. Previous pilot studies using VPA have showed conflicting results on the ability of VPA to reduce the size of HIV reservoirs. PURPOSE: As the role of VPA on HIV reservoirs remains unclear, we conducted a multicenter clinical trial with a specific study design to obtain optimal information on reservoir changes while exposing the smallest number of individuals to the experimental medication. METHOD: To this aim, a randomized, crossover design with 2 different treatment durations was implemented. By doubling the therapeutic period in one study arm, we were in a position to assess the impact of an extended duration of VPA on the size of the HIV reservoir and to evaluate the duration of treatment effects upon VPA withdrawal in the other arm. However, limitations for this type of study design included the logistical complexity of 2 uneven study arms and longer study duration. CONCLUSION: Despite the absence of demonstrable impact of VPA on reservoir size, such crossover study design should be considered in the early stage testing of novel HIV therapeutics targeted to reduce reservoir size or eradicate HIV.

Meeting report: Spontaneous lesions and diseases in wild, captive-bred, and zoo-housed nonhuman primates and in nonhuman primate species used in drug safety studies.

The combination of loss of habitat, human population encroachment, and increased demand of select nonhuman primates for biomedical research has significantly affected populations. There remains a need for knowledge and expertise in understanding background findings as related to the age, source, strain, and disease status of nonhuman primates. In particular, for safety/biomedical studies, a broader understanding and documentation of lesions would help clarify background from drug-related findings. A workshop and a minisymposium on spontaneous lesions and diseases in nonhuman primates were sponsored by the concurrent Annual Meetings of the American College of Veterinary Pathologists and the American Society for Veterinary Clinical Pathology held December 3-4, 2011, in Nashville, Tennessee. The first session had presentations from Drs Lowenstine and Montali, pathologists with extensive experience in wild and zoo populations of nonhuman primates, which was followed by presentations of 20 unique case reports of rare or newly observed spontaneous lesions in nonhuman primates (see online files for access to digital whole-slide images corresponding to each case report at http://www.scanscope.com/ACVP%20Slide%20Seminars/2011/Primate%20Pathology/view.apml). The minisymposium was composed of 5 nonhuman-primate researchers (Drs Bradley, Cline, Sasseville, Miller, Hutto) who concentrated on background and spontaneous lesions in nonhuman primates used in drug safety studies. Cynomolgus and rhesus macaques were emphasized, with some material presented on common marmosets. Congenital, acquired, inflammatory, and neoplastic changes were highlighed with a focus on clinical, macroscopic, and histopathologic findings that could confound the interpretation of drug safety studies.

Leukotrienes and isocyanate-induced asthma: a pilot study.

BACKGROUND: The role of leukotrienes (LTs) in the pathophysiology of isocyanate-induced asthma is not well known. OBJECTIVE: We sought to characterize the type of airway inflammation induced by exposure to isocyanates and to investigate whether exposure to isocyanates induced an increase in LT receptor cysteinyl leukotriene ((CysLT)(1), CysLT(2) and leukotriene B(4) receptor (BLT(1))) expression, as well as a release of LT (LTC(4) and leukotriene B(4) (LTB(4))) and IL-8 in both asthmatics with isocyanate-induced asthma and healthy subjects. METHODS: We investigated eight subjects with isocyanate-induced asthma and eight healthy subjects. Both groups underwent specific inhalation challenges to isocyanates in the laboratory. Induced sputum was collected before and after exposure to isocyanates. CysLT(1), CysLT(2) and BLT(1) expression was assessed by flow cytometry, whereas LTC(4), LTB(4) and IL-8 were measured in the sputum supernatants by enzyme immunoassay. RESULTS: Exposure to isocyanates induced an increase in sputum neutrophils only in subjects with occupational asthma. There was a significant increase in CysLT(1) and BLT(1) receptor expression, as well as a release of LTB(4) and IL-8 after exposure to isocyanates compared with the baseline, only in subjects with isocyanate-induced asthma, whereas there was no increase in LTC(4). Exposure to isocyanates did not induce any change in LT receptor expression nor in the levels of LTC(4), LTB(4) and IL-8, in healthy subjects. CONCLUSION: The neutrophilia observed after exposure to isocyanates is likely to be related to the release of LTB(4), probably enhanced by the increased expression of BLT(1) on neutrophils as well as by the release of IL-8. The significance of the increase of CysLT1 receptor expression on neutrophils is unknown and needs further investigation.

Anti-inflammatory activity of clarithromycin in adults with chronically inflamed sinus mucosa.

In a phase IV, open-label study, 25 patients with clinically stable chronic sinusitis and persistent maxillary sinus inflammation were treated for 14 days with clarithromycin 500 mg twice daily. Biopsy specimens of the maxillary sinus mucosa were obtained pretreatment and evaluated for macrophages (CD68), eosinophils (MBP), elastase, interleukin-6 (IL-6), IL-8, tumor necrosis factor-alpha (TNF-alpha), and activity of eosinophils (EG2), as well as edema score. Clinical signs and symptoms were assessed pretreatment, at the end of treatment, and 1 and 2 weeks later. Statistically significant reductions (P < or = .05) from pretreatment were observed for all markers of sinus mucosal inflammation, including CD68, EG2, elastase, IL-6, IL-8, TNF-alpha, and edema score, with a trend to decreased total eosinophil count. Improvement was observed for all clinical signs and symptoms of chronic sinusitis--sinus pain, sinus headache, nasal congestion, nasal discharge, and mucopurulent discharge--up to 14 days after the end of treatment. Cultures to evaluate persistent infection with Chlamydia pneumoniae showed negative results. Significant reductions in various markers of sinus mucosal inflammation support the role of clarithromycin in modulating immunologic responses. Improvement of clinical signs and symptoms in patients with chronic inflammatory sinusitis not meeting criteria for known or presumed bacterial infection was also noted up to 2 weeks after completion of a 14-day course of clarithromycin.

Expression of E- and P-selectin in tumor necrosis factor-induced dermatitis in dogs.

Adhesion molecules on endothelial cells play an important role in leukocyte recruitment in several inflammatory processes. Vascular selectins mediate the initial adhesion of leukocytes to the blood vessel wall during their extravasation into inflamed tissues, and in vitro studies in dogs have shown that selectin expression can be induced by cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1). The objective of this study was to determine whether vascular selectins are induced by cytokines in vivo in a cutaneous model of inflammation in dogs. Skin biopsies were collected from nine dogs at various time points after an intradermal injection of TNF-alpha (10 ng/site) or phosphate-buffered saline containing 0.1% bovine serum albumin, and immunohistochemistry was performed using anti-P-selectin (MD3) and anti-E-selectin (CL37) monoclonal antibodies. In all animals, TNF-alpha induced an inflammatory reaction that was maximal at 12 hours and then decreased by 24 and 48 hours. Control skin displayed no expression of E- and P-selectin, whereas TNF-alpha induced the expression of P-selectin and E-selectin on dermal vessels that was highest at 12 hours and 3 hours, respectively (P < 0.05). Numerous platelet aggregates recognized by the anti-P-selectin antibody were present in the lumina of vessels and in perivascular tissues. These results demonstrate that TNF-alpha can induce the expression of P- and E-selectin in vivo in dog skin and suggest that these selectins are involved in leukocyte recruitment in canine dermatitis.

Low- and high-density lipoprotein metabolism in HepG2 cells expressing various levels of apolipoprotein E.

To determine the importance of hepatic apolipoprotein (apo) E in lipoprotein metabolism, HepG2 cells were transfected with a constitutive expression vector (pRc/CMV) containing either the complete or the first 474 base pairs of the human apoE cDNA inserted in an antisense orientation, for apoE gene inactivation, or the full-length human apoE cDNA inserted in a sense orientation for overexpression of apoE. Stable transformants were obtained that expressed 15, 24, 226, and 287% the apoE level of control HepG2 cells. The metabolism of low-density lipoprotein (LDL) and high-density lipoprotein-3 (HDL(3)), two lipoprotein classes following both holoparticle and cholesteryl esters (CE)-selective uptake pathways, was compared between all these cells. LDL-protein degradation, an indicator of the holoparticle uptake, was greater in low apoE expressing cells than in control or high expressing cells, while HDL(3)-protein degradation paralleled the apoE levels of the cells (r(2) = 0.989). LDL- and HDL(3)-protein association was higher in low apoE expressing cells compared to control cells. In opposition, LDL- and HDL(3)-CE association was not different from control cells in low apoE expressing cells but rose in high apoE expressing cells. In consequence, the CE-selective uptake (CE/protein association ratio) was positively correlated with the level of apoE expression in all cells for both LDL (r(2) = 0.977) and HDL(3) (r(2) = 0.998). We also show that, although in normal and low apoE expressor cells, 92% of LDL- and 80% HDL(3)-CE hydrolysis is sensitive to chloroquine suggesting a pathway linked to lysosomes for both lipoproteins, cells overexpressing apoE lost 60% of chloroquine-sensitive HDL(3)-CE hydrolysis without affecting that of LDL-CE. Thus, the level of apoE expression in HepG2 cells determines the fate of LDL and HDL(3).

[Biological monitoring of exposure to polycyclic aromatic hydrocarbons among people living nearby an aluminum smelter in the province of Québec]. / Surveillance biologique de l'exposition aux hydrocarbures aromatiques polycycliques chez des personnes vivant aux environs d'une aluminerie québécoise.

BACKGROUND: The objective of the study is to estimate the exposure to pyrene, an indicator of polycyclic aromatic hydrocarbons (PAHs) in the general environment, by using the internal dose of 1-hydroxypyrene (1-OHP) among people living nearby an aluminium smelter in the Montérégie area, Province of Québec, Canada. METHODS: This cross-sectional study was done in November and December 1998. Participants were randomly selected according to their environmental exposure to PAHs and were distributed according to three exposure levels (high, low, none). Altogether, 121 non smokers aged between 18 to 70 years were recruited for the study. Persons occupationally exposed to PAHs or using tar-based medications were excluded. Those with personal medical conditions that affect the metabolism of pyrene were also excluded. Urine samples were taken in the morning and analysed by gas chromatography and mass spectrometry (GC-MS) to determine the concentration of the metabolite, 1-OHP. RESULTS: Among the exposed group (n=78), the geometric mean of urinary concentration of 1-OHP was 0.073 micromol/mol creatinine compared to 0.060 micromol/mol creatinine for the control group (n=40). The difference did not reach statistical significance (p=0.09). Geometric means among the three groups of exposure (high, low, none) were respectively 0.079, 0.067 and 0.060 micromol/mol creatinine (p=0.13). Accounting for personal risk factors, such as diet, passive smoke, use of wood heating and time spent at home during the three days prior to urine sampling, did not change previous results. CONCLUSION: This study indicates that the environmental levels of PAH produced by this factory are low and do not contribute significantly to the body burden of PAH as measured by 1-OHP.

Synovial sarcoma in an Ayrshire heifer.

An 8-month-old Ayrshire heifer had a rapidly growing mass in the axillary region of the left thoracic limb. The mass surrounded the distal humerus and entrapped nerves of the brachial plexus, causing an abnormal gait. Histologically, the mass was composed of clusters and cords of round to polygonal cells with scattered, spindle-shaped cells. The neoplastic cells stained positively for vimentin and cytokeratin. No staining was found with S-100 protein, kappa and lambda light chains, or T-cell markers by immunohistochemistry. On electron microscopic evaluation, the cytoplasm of the neoplastic cells contained few organelles, principally rough and smooth endoplasmic reticulum and mitochondria. This synovial sarcoma has histologic and ultrastructural features characteristic of the poorly differentiated subtype of synovial sarcoma in the human classification system.

Induction of prostaglandin G/H synthase-2 in a canine model of spontaneous prostatic adenocarcinoma.

BACKGROUND: Prostate cancer is the most frequently occurring cancer in men in the United States, with an estimated 179 300 new cases in 1999. The induction of prostaglandin G/H synthase (PGHS), a key rate-limiting enzyme in prostaglandin biosynthesis, has been implicated in various cancers, most notably in colorectal cancers; however, the induction of PGHS expression in prostate cancer in vivo has not been reported for any species. The dog is the only nonhuman species that frequently develops spontaneous cancer of the prostate with increasing age, and the objective of this study was to determine whether PGHS isoenzymes were expressed in canine prostatic adenocarcinomas. METHODS: Four normal canine prostatic tissues and 24 canine prostatic adenocarcinomas were studied by means of immunohistochemistry and immunoblot analysis, using polyclonal antibodies specific for each of the two PGHS isoenzymes, PGHS-1 and PGHS-2. All P values were obtained by use of two-sided Fisher's exact tests. RESULTS: PGHS-1 immunostaining was localized to stromal fibroblasts and vascular endothelium in normal and cancerous prostates. PGHS-2 was not detected in normal prostates, but it was expressed by epithelial tumor cells in 18 (75%) of the 24 adenocarcinomas (P =.01). Immunoblot analysis confirmed the presence of PGHS-1 (69 000 molecular weight) in normal and cancerous tissues and the expression of PGHS-2 (72 000- to 74 000-molecular-weight doublet) only in prostatic adenocarcinomas. CONCLUSION: To our knowledge, these results demonstrate for the first time that PGHS-2 is induced in the majority of canine spontaneous prostatic adenocarcinomas and suggest that its expression may be involved in prostate cancer.

Selective uptake of cholesteryl esters from various classes of lipoproteins by HepG2 cells.

Selective uptake of cholesteryl esters (CE) from lipoproteins by cells has been extensively studied with high density lipoproteins (HDL). It is only recently that such a mechanism has been attributed to intermediate and low density lipoproteins (IDL and LDL). Here, we compare the association of proteins and CE from very low density lipoproteins (VLDL), IDL, LDL and HDL3 to HepG2 cells. These lipoproteins were either labelled in proteins with 125I or in CE with 3H-cholesteryl oleate. We show that, at any lipoprotein concentration, protein association to the cells is significantly smaller for IDL, LDL, and HDL3 than CE association, but not for VLDL. At a concentration of 20 microg lipoprotein/mL, these associations reveal CE-selective uptake in the order of 2-, 4-, and 11-fold for IDL, LDL, and HDL3, respectively. These studies reveal that LDL and HDL3 are good selective donors of CE to HepG2 cells, while IDL is a poor donor and VLDL is not a donor. A significant inverse correlation (r2 = 0.973) was found between the total lipid/protein ratios of the four classes of lipoproteins and the extent of CE-selective uptake by HepG2 cells. The fate of 3H-CE of the two best CE donors (LDL and HDL3) was followed in HepG2 cells after 3 h of incubation. Cells were shown to hydrolyze approximately 25% of the 3H-CE of both lipoproteins. However, when the cells were treated with 100 microM of chloroquine, a lysosomotropic agent, 85 and 40% of 3H-CE hydrolysis was lost for LDL and HDL3, respectively. The fate of LDL and HDL3-CE in HepG2 cells deficient in LDL-receptor was found to be the same, indicating that the portion of CE hydrolysis sensitive to chloroquine is not significantly linked to LDL-receptor activity. Thus, in HepG2 cells, the magnitude of CE-selective uptake is inversely correlated with the total lipid/protein ratios of the lipoproteins and CE-selective uptake from the two best CE donors (LDL and HDL3) appears to follow different pathways.

Low density lipoprotein-receptor plays a major role in the binding of very low density lipoproteins and their remnants on HepG2 cells.

The binding to HepG2 cells of very low density lipoproteins (VLDL) and their remnants (IDL) was alternatively, in the past, attributed to the low density lipoprotein receptor (LDLr) or to an apoE-specific receptor. In order to resolve this issue, we have compared the binding of those lipoproteins labelled with iodine-125 to normal and LDLr deficient HepG2 cells. Those deficient cells were obtained by a constitutive antisense strategy and their LDLr level is 14% the level of normal HepG2 cells. By saturation curve analysis, we show that VLDL and IDL bind to high and low affinity sites on cells. The low affinity binding was eliminated by conducting the assay in presence of a 200-fold excess of HDL3 respective to the concentrations of 125I-labelled VLDL and IDL. For 125I-VLDL high affinity binding to normal HepG2 cells, we found a dissociation constant (Kd) of 21.2 +/- 3.7 micrograms prot./ml (S.E., N = 5) and a maximal binding capacity (Bmax) of 0.0312 +/- 0.0063 microgram prot./mg cell prot, while we have measured a Kd of 5.3 +/- 0.8 and a Bmax of 0.0081 +/- 0.0014 with LDLr deficient cells. This indicates that LDLr is responsible for 74% of VLDL binding to HepG2 cells and that the non-LDLr high affinity receptor has a higher affinity for VLDL than LDLr. A 53% loss of 125I-IDL binding capacity was measured with LDLr deficient cells compared with normal cells (Bmax: 0.028 +/- 0.005 versus 0.059 +/- 0.006), while no significant statistical difference was found between affinities. The study shows that the LDLr is almost the only contributor in VLDL binding, while it shares IDL binding capacity with another high affinity receptor. The physiological importance of LDLr is confirmed by an almost equivalent loss of IDL and VLDL degradation in LDLr deficient cells.

How effective are supplementary doses of opioids for dyspnea in terminally ill cancer patients? A randomized continuous sequential clinical trial.

Supplementary doses of opioids are recommended to relieve dyspnea in terminally ill cancer patients. We conducted a randomized continuous sequential clinical trial to evaluate their efficacy. We recruited 33 terminally ill cancer patients from three palliative care centers, all of whom had persistent dyspnea after rest and treatment with oxygen. Patients formed 15 successive pairs matched on route of administration. Within each pair, the order of allocation was randomly assigned, one patient receiving 25%, the other 50% of his 4-hourly opioid dose. Five measurements of dyspnea intensity and respiratory frequency were made during 4 hours of follow-up. For each pair, a preference was attributed to the more effective regimen. The two regimens received an almost equal number of paired preferences (8 vs. 7). Overall, both mean dyspnea intensity and respiratory frequency decreased significantly relative to baseline. Dyspnea reduction was relatively greater in patients with initially low and moderate dyspnea intensity. In terminally ill cancer patients with persistent dyspnea, 25% of the equivalent 4-hourly dose of opioid may be sufficient to reduce both dyspnea intensity and tachypnea for 4 hours.

Uptake and fate of class B scavenger receptor ligands in HepG2 cells.

Class B scavenger receptors (SR-Bs) interact with native, acetylated and oxidized low-density lipoprotein (LDL, AcLDL and OxLDL), high-density lipoprotein (HDL3) and maleylated BSA (M-BSA). The aim of this study was to analyze the catabolism of CD36- and LIMPII-analogous-1 (CLA-1), the human orthologue for the scavenger receptor class B type I (SR-BI), and CD36 ligands in HepG2 (human hepatoma) cells. Saturation binding experiments revealed moderate-affinity binding sites for all the SR-B ligands tested with dissociation constants ranging from 20 to 30 microg.mL-1. Competition binding studies at 4 degrees C showed that HDL and modified and native LDL share common binding site(s), as OxLDL competed for the binding of 125I-LDL and 125I-HDL3 and vice versa, and that only M-BSA and LDL may have distinct binding sites. Degradation/association ratios for SR-B ligands show that LDL is very efficiently degraded, while M-BSA and HDL3 are poorly degraded. The modified LDL degradation/association ratio is equivalent to 60% of the LDL degradation ratio, but is three times higher than that of HDL3. All lipoproteins were good cholesteryl ester (CE) donors to HepG2 cells, as a 3.6-4.7-fold CE-selective uptake ([3H]CE association/125I-protein association) was measured. M-BSA efficiently competed for the CE-selective uptake of LDL-, OxLDL-, AcLDL- and HDL3-CE. All other lipoproteins tested were also good competitors with some minor variations. Hydrolysis of [3H]CE-lipoproteins in the presence of chloroquine demonstrated that modified and native LDL-CE were mainly hydrolyzed in lysosomes, whereas HDL3-CE was hydrolyzed in both lysosomal and extralysosomal compartments. Inhibition of the selective uptake of CE from HDL and native modified LDL by SR-B ligands clearly suggests that CLA-1 and/or CD36 are involved at least partially in this process in HepG2 cells.

A pilot study on upper esophageal sphincter dilatation for the treatment of dysphagia in patients with oculopharyngeal muscular dystrophy.

Upper esophageal sphincter (UES) dilatation was done for the treatment of moderate to severe dysphagia with a Maloney bougie in 14 patients with oculopharyngeal muscular dystrophy (OPMD) or with an achalasia dilator in three patients. The severity of dysphagia prior to UES dilatation was evaluated by a 15-point dysphagia score, a pharyngeal and esophageal manometry and a radionuclide pharyngoesophageal transit study. Using actuarial life table, the improvement rate after dilatation with Maloney bougie was 64.3% (95% CI 39.2-89.4) at 3- and 6-month follow-ups, and was 55.7% (95% CI 28.9-82.5) at 12- and 18-month follow-ups. At 3-month post-dilatation, we observed a significant reduction of the mean dysphagia score from 9.6 to 7.2 (P = 0.05). No significant manometric or radionuclide factors were found to predict effective dilatation. The results of this pilot study showed that UES dilatation with Maloney bougie or achalasia dilator may be an effective treatment of moderate dysphagia in patients with OPMD. However, further studies with larger sample sizes are needed to corroborate these results and to assess long-term outcome.

Structural and evolutionary relationships among chitinases of flowering plants.

The analysis of nuclear-encoded chitinase sequences from various angiosperms has allowed the categorization of the chitinases into discrete classes. Nucleotide sequences of their catalytic domains were compared in this study to investigate the evolutionary relationships between chitinase classes. The functionally distinct class III chitinases appear to be more closely related to fungal enzymes involved in morphogenesis than to other plant chitinases. The ordering of other plant chitinases into additional classes mainly relied on the presence of auxiliary domains-namely, a chitin-binding domain and a carboxy-terminal extension-flanking the main catalytic domain. The results of our phylogenetic analyses showed that classes I and IV form discrete and well-supported monophyletic groups derived from a common ancestral sequence that predates the divergence of dicots and monocots. In contrast, other sequences included in classes I* and II, lacking one or both types of auxiliary domains, were nested within class I sequences, indicating that they have a polyphyletic origin. According to phylogenetic analyses and the calculation of evolutionary rates, these chitinases probably arose from different class I lineages by relatively recent deletion events. The occurrence of such evolutionary trends in cultivated plants and their potential involvement in host-pathogen interactions are discussed.