Peptide IC-20, encoded by skin kininogen-1 of the European yellow-bellied toad, Bombina variegata, antagonizes bradykinin-induced arterial smooth muscle relaxation

The objectives were to determine if the skin secretion of the European yellow-bellied toad (Bombina variegata), in common with other related species, contains a bradykinin inhibitor peptide and to isolate and structurally characterize this peptide. Materials and Methods: Lyophilized skin secretion obtained from this toad was subjected to reverse phase HPLC fractionation with subsequent bioassay of fractions for antagonism of the bradykinin activity using an isolated rat tail artery smooth muscle preparation. Subsequently, the primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy, and molecular cloning, following which a synthetic replicate was chemically synthesised for bioassay. Results: A single peptide of molecular mass 2300.92 Da was resolved in HPLC fractions of skin secretion and its primary structure determined as IYNAIWP-KH-NK-KPGLL-. Database interrogation with this sequence indicated that this peptide was encoded by skin kininogen-1 previously cloned from B. variegata. The blank cycles were occupied by cysteinyl (C) residues and the peptide was located toward the C-terminus of the skin kininogen, and flanked N-terminally by a classical -KR- propeptide convertase processing site. The peptide was named IC-20 in accordance (I = N-terminal isoleucine, C = C-terminal cysteine, 20 = number of residues). Like the natural peptide, its synthetic replicate displayed an antagonism of bradykinin-induced arterial smooth muscle relaxation. Conclusion: IC-20 represents a novel bradykinin antagonizing peptide from amphibian skin secretions and is the third such peptide found to be co-encoded with bradykinins within skin kininogens.

Piperonyl butoxide enhances triclabendazole action against triclabendazole-resistant Fasciola hepatica.

A study has been carried out to determine whether the action of triclabendazole (TCBZ) against the liver fluke, Fasciola hepatica is altered by inhibition of the cytochrome P450 (CYP 450)-mediated drug metabolism pathway. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for these experiments, the basic design of which is given in the paper by Devine et al. (2010a). Piperonyl butoxide (PB) was the CYP P450 inhibitor used. Morphological changes resulting from drug treatment and following metabolic inhibition were assessed by means of transmission electron microscopy. After treatment with either TCBZ or TCBZ.SO on their own, there was greater disruption to the TCBZ-susceptible than TCBZ-resistant isolate. However, co-incubation with PB+TCBZ, but more particularly PB+TCBZ.SO, led to greater changes to the TCBZ-resistant isolate than with each drug on its own, with blebbing of the apical plasma membrane, severe swelling of the basal infolds and their associated mucopolysaccharide masses in the syncytium and flooding in the internal tissues. Golgi complexes were greatly reduced or absent in the tegumental cells and the synthesis and production of secretory bodies were badly disrupted. The mitochondria were swollen throughout the tegumental system and the somatic muscle blocks were disrupted. With the TCBZ-susceptible Cullompton isolate, there was a limited increase in drug action following co-incubation with PB. The results provide evidence that the condition of a TCBZ-resistant fluke can be altered by inhibition of drug metabolism. Moreover, they support the concept that altered drug metabolism contributes to the mechanism of resistance to TCBZ

Early target cells of measles virus after aerosol infection of non-human primates

Measles virus (MV) is highly infectious, and has long been thought to enter the host by infecting epithelial cells of the respiratory tract. However, epithelial cells do not express signaling lymphocyte activation molecule (CD150), which is the high-affinity cellular receptor for wild-type MV strains. We have generated a new recombinant MV strain expressing enhanced green fluorescent protein (EGFP), based on a wild-type genotype B3 virus isolate from Khartoum, Sudan (KS). Cynomolgus macaques were infected with a high dose of rMV(KS)EGFP by aerosol inhalation to ensure that the virus could reach the full range of potential target cells throughout the entire respiratory tract. Animals were euthanized 2, 3, 4 or 5 days post-infection (d.p.i., n?=?3 per time point) and infected (EGFP(+)) cells were identified at all four time points, albeit at low levels 2 and 3 d.p.i. At these earliest time points, MV-infected cells were exclusively detected in the lungs by fluorescence microscopy, histopathology and/or virus isolation from broncho-alveolar lavage cells. On 2 d.p.i., EGFP(+) cells were phenotypically typed as large mononuclear cells present in the alveolar lumen or lining the alveolar epithelium. One to two days later, larger clusters of MV-infected cells were detected in bronchus-associated lymphoid tissue (BALT) and in the tracheo-bronchial lymph nodes. From 4 d.p.i. onward, MV-infected cells were detected in peripheral blood and various lymphoid tissues. In spite of the possibility for the aerosolized virus to infect cells and lymphoid tissues of the upper respiratory tract, MV-infected cells were not detected in either the tonsils or the adenoids until after onset of viremia. These data strongly suggest that in our model MV entered the host at the alveolar level by infecting macrophages or dendritic cells, which traffic the virus to BALT or regional lymph nodes, resulting in local amplification and subsequent systemic dissemination by viremia.

Fasciola hepatica: time-dependent disruption of spermatogenesis following in vivo treatment with triclabendazole

Sheep infected with the Cullompton isolate of Fasciola hepatica were treated with triclabendazole at a concentration of 10 mg/kg at 12 weeks post-infection. Adult flukes were recovered from the liver and, where present, from the gall bladder at 48, 72 and 96 h post-treatment (pt). Gross changes to the spermatogenic cells of the testis were examined by histology and ultrastructural alterations were visualised via transmission electron microscopy. Disruption was progressive in nature, with the testis tubules becoming shrunken, vacuolated and gradually more denuded of cellular content over the 96-h time period. From 48 h pt, the number of primary and secondary spermatogonia decreased and multinucleate spermatogonial cells were frequent. Later, developmental stages were uncommon, giving rise to much empty space within the tubules. By 72 h pt, the tubules contained many apoptotic and degraded cells and had an extremely disorganised appearance. At 96 h pt, the tubules were almost completely empty, with the exception of the remains of degraded spermatogenic cells. These results indicate that triclabendazole severely disrupts spermatogenesis in the liver fluke from 48 h pt in vivo.

Fasciola hepatica: Comparative effects of host resistance and parasite intra-specific interactions on size and reproductive histology in flukes from rats infected with isolates differing in triclabendazole sensitivity

The efficacies of putative fasciolicides and vaccines against Fasciola hepatica are frequently monitored in clinical and field trials by determination of fluke egg output in host faeces and by worm counts in the host liver at autopsy. Less often used are parameters based on fluke size and histology, yet these can provide important indications of specific effects on the development of particular germ-line or somatic tissues, especially in relation to the timing and profligacy of egg production. In this study. F. hepatica metacercariae of two distinct isolates, the triclabendazole (TCBZ)-sensitive Cullompton isolate and the TCBZ-resistant Oberon isolate, were administered to rats as single-isolate or mixed-isolate infections. At autopsy 16 weeks later individual adult flukes were counted, measured and the reproductive organs were examined histologically. The degree of development of the testis tubules in each fluke was represented by a numerical score, based on the proportion of the histological section profiles occupied by testis tissue. The level of anti-F. hepatica antibody in the serum of each rat was determined by ELISA. It was found that Cullompton flukes were significantly larger than Oberon flukes, and that significantly more Cullompton metacercariae developed to adults than Oberon metacercariae. The Cullompton flukes showed histological evidence of aspermy and spermatogenic arrest, which was reflected in quantitatively reduced testicular development, as compared with the Oberon isolate. In Cullompton flukes, parthenogenetic egg development is implied. The size of Cullompton and Oberon flukes was significantly related to the number of adult flukes recovered, to the number of metacercariae administered, and to the percentage success of infection. The testis development score in both isolates was significantly related to the number of adult flukes recovered but not to the number of metacercariae administered, or to the percentage success of infection. Fluke size was positively related to testis score for both isolates, and a significant negative relationship was found between percentage success of infection and metacercarial dose. The results are interpreted in terms of differing interactions between various numbers of young flukes and host immunity during invasion of and migration in the hepatic parenchyma, and of fluke intra-specific (possibly pheromonal) stimulatory effects in the final stages of development, within the host bile ducts. No significant relationships were found between host antibody levels and fluke size or testis score. False positive serological reactions were found in some rats that had been infected, but found to harbour no flukes at autopsy. Clearly the act of eliminating the flukes involved generation of an immune response. (C) 2011 Elsevier B.V. All rights reserved.

Disruption of egg formation by Fasciola hepatica following treatment in vivo with triclabendazole in the sheep host

Eight indoor-reared cross-bred sheep with no prior exposure to Fasciola hepatica were infected by oral gavage with 200 metacercarial cysts of the triclabendazole (TCBZ)-susceptible Cullompton isolate of F. hepatica. Twelve weeks after infection, sheep were treated with 10 mg/kg triclabendazole. Two sheep were euthanised per time period; at 48 h, 72 h and 96 h post-treatment (pt). Two untreated control sheep were euthanised at 96 h pt. Flukes were recovered from the liver and, if present, from the gall bladder of the sheep. They were processed for whole mount analysis, histology and transmission electron microscopy of the female reproductive system; specifically, the uterus, vitelline follicles. Mehlis' gland and ovary.

Development of an egg hatch assay for the diagnosis of triclabendazole resistance in Fasciola hepatica: proof of concept.

The aim of this study was to develop an Egg Hatch Assay (EHA) test for the detection of triclabendazole (TCBZ) resistance in Fasciola hepatica. A number of fluke isolates were used, of differing sensitivity to TCBZ. Eggs were exposed to solutions of triclabendazole sulphoxide (TCBZ.SO) for 14 days, then triggered to hatch. Egg development was divided into 6 distinct and easily identifiable stages: dead, empty, unembryonated, cell division, eye spot and hatched. The number of eggs reaching those stages was recorded. Initially, the discriminating dose (1% hatch) was determined for the Cullompton isolate, used as TCBZ-susceptible (TCBZ-S) standard. Once this concentration had been resolved, the response of different isolates to this concentration was examined. The hatch rate of the Fairhurst isolate was not significantly different from that of the Cullompton isolate, confirming its TCBZ-S status. The Patagonia isolate has not been exposed to TCBZ in the field and should be TCBZ-S: the results of the EHA supported this. The egg hatch response of the Oberon and Dutch isolates differed significantly from that of the Cullompton isolate; the former isolates are regarded as TCBZ-resistant (TCBZ-R) and the results confirmed this. Another isolate, the Leon isolate, was originally described as being TCBZ-R, but has since been shown to be TCBZ-S. There was no difference in its response to TCBZ.SO in the EHA from the Cullompton (and Fairhurst and Patagonia) isolate(s), further indicating its TCBZ-S status. The impact of TCBZ.SO treatment on the component stages of egg development was determined and revealed differences between the isolates. In conclusion, the results of the study have shown that it is possible to discriminate between TCBZ-S and TCBZ-R isolates of F. hepatica on the basis of the response of their eggs to an EHA and the test could be used to evaluate the TCBZ sensitivity of unknown field isolates

Production and Evaluation of Antibodies and Phage Display-Derived Peptide Ligands for Immunomagnetic Separation of Mycobacterium bovis

This study describes the development and optimization of an immunomagnetic separation (IMS) method to isolate Mycobacterium bovis cells from lymph node tissues. Gamma-irradiated whole M. bovis AF2122/97 cells and ethanol-extracted surface antigens of such cells were used to produce M. bovis-speci?c polyclonal and monoclonal antibodies in rabbits and mice. They were also used to generate M. bovis-speci?c peptide ligands by phage display biopanning. The various antibodies and peptide ligands obtained were used to coat MyOne tosyl-activated Dynabeads (Life Technologies), singly or in combination, and evaluated for IMS. Initially, M. bovis capture from Middlebrook 7H9 broth suspensions (concentration range, 10 to 10⁵ CFU/ml) was evaluated by IMS combined with an M. bovis-speci?c touchdown PCR. IMS-PCR results and, subsequently, IMS-culture results indicated that the beads with greatest immunocapture capability for M. bovis in broth were those coated simultaneously with a monoclonal antibody and a biotinylated 12-mer peptide. These dually coated beads exhibited minimal capture (mean of 0.36% recovery) of 12 other Mycobacterium spp. occasionally encountered in veterinary tuberculosis (TB) diagnostic laboratories. When the optimized IMS method was applied to various M. bovis-spiked lymph node matrices, it demonstrated excellent detection sensitivities (50% limits of detection of 3.16 and 57.7 CFU/ml of lymph node tissue homogenate for IMS-PCR and IMS-culture, respectively). The optimized IMS method therefore has the potential to improve isolation of M. bovis from lymph nodes and hence the diagnosis of bovine tuberculosis.

In vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo

Respiratory syncytial virus (RSV) is the major viral cause of severe pulmonary disease in young infants worldwide. However, the mechanisms by which RSV causes disease in humans remain poorly understood. To help bridge this gap, we developed an ex vivo/in vitro model of RSV infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs), the primary targets of RSV infection in vivo. Our RSV/WD-PBEC model demonstrated remarkable similarities to hallmarks of RSV infection in infant lungs. These hallmarks included restriction of infection to noncontiguous or small clumps of apical ciliated and occasional nonciliated epithelial cells, apoptosis and sloughing of apical epithelial cells, occasional syncytium formation, goblet cell hyperplasia/metaplasia, and mucus hypersecretion. RSV was shed exclusively from the apical surface at titers consistent with those in airway aspirates from hospitalized infants. Furthermore, secretion of proinflammatory chemokines such as CXCL10, CCL5, IL-6, and CXCL8 reflected those chemokines present in airway aspirates. Interestingly, a recent RSV clinical isolate induced more cytopathogenesis than the prototypic A2 strain. Our findings indicate that this RSV/WD-PBEC model provides an authentic surrogate for RSV infection of airway epithelium in vivo. As such, this model may provide insights into RSV pathogenesis in humans that ultimately lead to successful RSV vaccines or therapeutics.

Standardisation and comparison of methods employed for microbial cell surface hydrophobicity and charge determination

Whilst there are a number of methods available to characterise the cell surface hydrophobicity (CSH) and cell surface charge (CSC) of microorganisms, there is still debate concerning the correlation of results between individual methods. In this study, the techniques of bacterial adherence to hydrocarbons (BATH) and hydrophobic interaction chromatography (HTC) were used to measure CSH. Electrostatic interaction chromatography (ESIC) and zeta potential (ZP) measurements were used to determine CSC. To allow meaningful comparisons between the BATH and HIC tests, between ESIC and ZP and also between CSH and CSC, the buffer systems employed in each test were standardised (phosphate buffered saline, pH 7.3, 0.01 mM). Isolates of Staphylococcus epidermidis derived from microbial biofilm were used as the test organism in this study. The isolates examined exhibited primarily medium to high CSH and a highly negative CSC. Good correlation of CSH measurement was observed between the BATH and HIC tests (r = 0.89). Good correlation was observed between ESIC (anionic exchange column) and ZP measurements. No correlations were observed between isolate CSC and either increased or decreased CSH. It is recommended that whenever comparisons of various methods to determine either CSC or CSH (by partitioning methods), the buffer systems should remain constant throughout to achieve consistency of results.

Early onset of changes to the reproductive system of Fasciola hepatica following in vivo treatment with triclabendazole

Lambs infected with the Cullompton triclabendazole (ICBZ)-susceptible isolate of Fasciola hepatica were treated with TCBZ at a dosage of 10 mg/kg at 16 weeks post-infection. Adult flukes were recovered from the liver at 3 h, 24 h, 48 h and 60 h post-treatment (pt). They were processed for histological analysis of the uterus, Mehlis' gland, vitellaria, ovary and testis. At 3 h pt, the flukes were essentially similar to the controls and were producing normal eggs. Egg production had ceased by 24 h pt. At this time period, the cells of the Mehlis' gland showed some evidence of vacuolation, but otherwise were relatively normal. A shift in the population of vitelline cells towards mature cells was observed at 24 h pt, and this trend continued at later time-periods. It was accompanied by a breakdown of the cells and the presence of apoptotic bodies. Marked changes to the ovary were first noted at 48 h Pt, as evidenced by vacuolation and the presence of apoptotic bodies. Some disruption to the testis was seen at 24 h pt, with a reduction in the population of spermatogenic cells, the appearance of apoptotic bodies and some peripheral vacuolation of the tubules. These abnormalities increased in severity with longer time periods pt. The results bring forward the time-line of cessation of egg production by 24 h, demonstrating that this process is affected very rapidly pt. (C) 2011 Elsevier B.V. All rights reserved.

Life history variation in Dasya ocellata (Dasyaceae, Rhodophyta)

Four of the five members of the Dasyaceae found in the British Isles, Dasya corymbifera J. Agardh, Dasya hutchinsiae Harvey, Dasya punicea Meneghini ex Zanardini and Heterosiphonia plumosa (Ellis) Batters, appear to have Polysiphonia-type life histories on the basis of evidence from field collections of tetrasporophytes and gametophytes. In collections from the British Isles of the fifth species, Dasya ocellata (Grateloup) Harvey, only tetrasporophytes have ever been observed, but there are two reports of gametophytes in this species from further south in Europe. Dasya ocellata tetraspores were isolated into culture from populations in Strangford Lough, Northern Ireland, and Agadir, Morocco, where one female thallus was collected amongst tetrasporophytes. Dasya ocellata from Ireland underwent a direct tetraspore-to-tetrasporophyte life history, which was followed through two complete cycles. Karyological studies showed that meiosis does not occur during tetrasporangial development: tetrasporangia are mitotic, with c. 64 small chromosomes. Comparison with chromosome numbers in meiotic tetrasporangia of D. hutchinsiae (n = c. 32) showed that this is the diploid chromosome complement. Tetraspores from the Moroccan isolate, by contrast, gave rise to gametophytes (although only the males became fertile) and tetrasporophyte recycling did not occur. Thalli sampled from a population in southern Portugal consisted only of tetrasporophytes. Dasya ocellata, like many members of the Ceramiales, shows intraspecific life history variability; a sexual life history apparently occurs only in southern populations.

A MOLECULAR AND MORPHOLOGICAL ANALYSIS OF THE GYMNOGONGRUS-DEVONIENSIS (RHODOPHYTA) COMPLEX IN THE NORTH-ATLANTIC

The Gymnogongrus devoniensis (Greville) Schotter complex in the North Atlantic Ocean was elucidated by comparative molecular, morphological, and culture studies. Restriction fragment length patterns and hybridization data on organellar DNA revealed two distinct taxa in samples from Europe and eastern Canada. Nucleotide sequences for the intergenic spacer between the large and small subunit genes of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and the adjoining regions of both genes, differed by 12.5-13.4% between the two taxa. One of the taxa, which included material from the type locality of G. devoniensis at Torbay, Devon, England, was taken to represent authentic G. devoniensis. Within this taxon, samples from Ireland, England, northern France, northern Spain, and southern Portugal showed great morphological variation, particularly in habit, but their Rubisco spacer sequences were identical or differed by only a single nucleotide. Constant morphological features included the development, from a single auxiliary cell, of the spherical cystocarp with a thick mucilage sheath that appears to be typical of Gymnogongrus species with internal cystocarps. Two life-history types were found. Northern isolates underwent a direct-type life history, recycling apomictic females by carpospores, whereas the Portuguese isolate followed a heteromorphic life history in which carpospores gave rise to a crustose tetrasporophyte.

Structural characterisation and pharmacology of KHEYLRFamide (AF2) and KSAYMRFamide (PF3/AF8) from Haemonchus contortus

The FMRFamide-related peptides (FaRPs), KHEYLRFamide (AF2) and KSAYMRFamide (PF3) were structurally characterised from the parasitic nematode of sheep, Haemonchus contortus (MH isolate). Both peptides were sequenced in a single gas-phase sequencing run and their structure confirmed by mass spectrometry which identified peptides of 920 Da (C-terminally amidated AF2) and 902/918 Da (C-terminally amidated non-oxidised/oxidised PF3, respectively). AF2 had inhibitory effects on H. contortus muscle and inhibited acetylcholine (ACh, 10 mu M)-induced contractions, with a threshold for activity of I mu M. PF3 induced concentration-dependent contractions of H. contortus (activity threshold, 10 nM) and enhanced ACh contractions. Compared with the MH isolate, an isolate of H. contortus which has reduced sensitivity to cholinergic drugs (Lawes isolate) was less sensitive to the effects of PF3. The concentration-response curves for the cholinergic compounds ACh and levamisole (LEV), and PF3, but not a control, KPNFIRFamide (PF4), showed a statistically similar shift. This study implicates PF3 in the modulation of cholinergic function in H. contortus. (C) 1999 Elsevier Science B.V. All rights reserved.

THE FMRFAMIDE-LIKE NEUROPEPTIDE AF2 (ASCARIS-SUUM) IS PRESENT IN THE FREE-LIVING NEMATODE, PANAGRELLUS-REDIVIVUS (NEMATODA, RHABDITIDA)

Available primary structural information suggests that the FMRFamide-related peptides (FaRPs) from parasitic and free-living nematodes are different, and that free-living forms may not represent appropriate models for the study of the neurochemistry of parasitic forms in the laboratory. However, here we report the isolation and unequivocal identification of AF2 (originally isolated from the parasite, Ascaris suum) from acidified alcoholic extracts of the free-living species, Panagrellus redivivus. While reverse-phase HPLC analysis of extracts revealed FMRFamide-immunoreactivity to be highly heterogeneous, AF2 was the predominant FMRFamide-immunoreactive peptide present (at least 26 pmol/g wet weight of worms). This peptide was also the major immunoreactant identified by an antiserum raised to the conserved C-terminal hexapeptide amide of mammalian pancreatic polypeptide (PP), which has been used previously to isolate neuropeptide F (NPF). These observations were confirmed by radioimmunoassay and chromatographic fractionation of an acidified alcoholic extract of A. suum heads. The FMRFamide-related peptides present in a nematode extract may be highly dependent on the extraction medium employed, and these data would suggest that this complement of neuropeptides may not be as different between parasitic and free-living nematodes as initial studies have suggested. Finally, all of the evidence suggests that NPF is not present in nematodes and that the PP-immunoreactant previously demonstrated immunochemically is probably AF2.