Alpha-1 antitrypsin limits neutrophil extracellular trap disruption of airway epithelial barrier function.

Neutrophil extracellular traps contribute to lung injury in cystic fibrosis and asthma, but the mechanisms are poorly understood. We sought to understand the impact of human NETs on barrier function in primary human bronchial epithelial and a human airway epithelial cell line. We demonstrate that NETs disrupt airway epithelial barrier function by decreasing transepithelial electrical resistance and increasing paracellular flux, partially by NET-induced airway cell apoptosis. NETs selectively impact the expression of tight junction genes claudins 4, 8 and 11. Bronchial epithelia exposed to NETs demonstrate visible gaps in E-cadherin staining, a decrease in full-length E-cadherin protein and the appearance of cleaved E-cadherin peptides. Pretreatment of NETs with alpha-1 antitrypsin (A1AT) inhibits NET serine protease activity, limits E-cadherin cleavage, decreases bronchial cell apoptosis and preserves epithelial integrity. In conclusion, NETs disrupt human airway epithelial barrier function through bronchial cell death and degradation of E-cadherin, which are limited by exogenous A1AT.

Correction: Development of highly active anti-Pneumocystis bisbenzamidines: insight into the influence of selected substituents on the in vitro activity.

[This corrects the article DOI: 10.1039/C7MD00445A.].

Development of highly active anti-Pneumocystis bisbenzamidines: insight into the influence of selected substituents on the in vitro activity.

Here we describe the potency of 21 pentamidine analogues against the fungal pathogen, Pneumocystis carinii, in an ATP bioluminescent assay with toxicity profiles in 2 mammalian cell lines. Reduction of two 5-methyl-1,2,4-oxadiazole rings was applied to the synthesis of acid-labile bisamidines. Anti-Pneumocystis activity is discussed in the context of 3 groups of compounds depending on the main structural changes of the pentamidine lead structure. The groups include: 1) 1,4-bis(methylene)piperazine derivatives 1-5; 2) alkanediamide derivatives 6-10; 3) alkane-derived bisbenzamidines 11-21. IC50 values of 18 compounds were lower than the IC50 of pentamidine. Four bisamidines were active at nanogram concentrations. Introduction of sulfur atoms in the alkane bridge, replacement of the amidino groups with imidazoline rings, or attachment of nitro or amino groups to the benzene rings is responsible for remarkable activity of the new leading structures. The vast majority of compounds, including four highly active ones, can be classified as mild or nontoxic to host cells. These compounds show promise as candidates for new anti-Pneumocystis agents.

Development of a real-time reverse-transcription PCR for the detection and simultaneous pathotyping of Newcastle disease virus isolates using a novel probe.

A real-time reverse-transcription PCR (RRT-PCR) was developed to detect and pathotype avian paramyxovirus type 1 (APMV-1), also known as Newcastle disease virus (NDV), which had been grown in embryonated fowls' eggs. Two pairs of probes, VRP1 with ARP1 and VRP2 with ARP2, each with either the 'universal base' 2' deoxyinosine incorporated or both inosines and locked nucleic acids (LNAs) incorporated, were designed to detect, respectively, a diverse range of virulent and avirulent viral templates that included the region coding for the fusion protein cleavage site. Oligonucleotide VRP1 hybridised with 76 of the 84 virulent isolates tested, while VRP2 detected 82, including 17 isolates with five or six template-probe mismatches. An alternative conventional probe, VRP3, with no inosine bases or LNAs, failed to hybridise 7 of 13 isolates, all of which tested positive with VRP2. Real-time assays with ARP1 showed that it detected 21 of the 28 avirulent isolates tested, and ARP2 detected 22/28, including one present in a mixture with virulent NDV. Neither probe was able to detect those isolates that were classified in genogroup six. All probes were specific for detecting either virulent or avirulent NDV. A specific PCR fragment of the predicted size was obtained, using the primer set designed for this study, with the 112 NDV isolates tested, including those in genogroup six. This assay demonstrates a rapid means for simultaneous detection and pathotyping of notifiable avian disease due to NDV.

Partial characterisation of five cloned viruses differing in pathogenicity, obtained from a single isolate of pigeon paramyxovirus type 1 (PPMV-1) following passage in fowls' eggs.

Viruses with intracerebral pathogenicity indices (ICPIs) of 0.025, 0.55, 1.013 and 1.3. were cloned from a PPMV-1 isolate with an ICPI of 0.32 by passage in embryonated fowls' eggs. Deduced amino acid sequences of the haemagglutinin-neuraminidase (HN) and precursor fusion proteins (F0) showed them to have only a single amino acid difference: those with an ICPI value <0.7 had proline at amino acid position 453 of the F0 protein, and those with an ICPI value >0.7 contained a serine. The virus with an ICPI of 0.025 was further passaged, and the ICPI of non-cloned virus increased to 0.76/0.79, which was then reduced to 0.49 on cloning. The proline at residue 453 was retained, but there were two nucleotide changes in the virus of ICPI 0.49, T --> C at position 1769 in the untranslated region of the fusion gene and G --> A at position 437 of the HN gene, resulting in the amino acid change G --> R at position 116 in the HN protein.

Rapid in vitro assessment of the virulence of Newcastle disease virus isolates using the ligase chain reaction.

The ligase chain reaction was used to assess the virulence of isolates of Newcastle disease virus. In the main study, 18/18 virulent isolates whose nucleotide sequences that code for the cleavage site and fusor peptide regions were known, successfully ligated oligonucleotides in a primer mix for virulent viruses termed VPM. Five of these isolates yielded a more intense ligated product with a second primer mix for virulent viruses called VPM1. No ligation was evident with eight avirulent isolates in tests with VPM or VPM1, however, each of these viruses did yield a strong ligated product with the primer mix for avirulent viruses (AVPM) as did one virulent isolate considered to be a mixture. Two virulent Australian isolates, 1238/1998 and 1248/1998, showed low but seemingly specific ligation with AVPM. In a blind study, 8/9 virulent isolates whose sequences were unknown ligated primers in VPM. Three avirulent and one virulent isolate, the latter again probably a mixture, ligated primers in AVPM. Ligation of oligonucleotides in VPM and AVPM was detectable in mixtures where virulent and avirulent isolates represented 0.1% and 0.01% by volume respectively of the viral population. The results indicate that LCR offers a potential in vitro alternative to current in vivo tests for virulence determination of Newcastle disease virus isolates.

Rapid pathotyping of Newcastle disease virus using a single-chain Fv displayed on phage against the C-terminal end of the F2 polypeptide.

Filamentous bacteriophage display technology has been used to generate specific antibody fragments for differentiating virulent and avirulent Newcastle disease virus. A single-chain Fv fragment to the motif (112)RRQ(114), present at the F2 C-terminal end of many virulent Newcastle disease virus isolates, was isolated from a phage display library derived from a rabbit immunized with a peptide conjugate. An ELISA evaluation was carried out to test its ability to differentiate between 11 avirulent and 34 virulent NDV isolates. The antibody fragment reacted with 25/28 virulent viruses with the putative motif (112)RRQ(114). The three exceptions were viruses with an arginine instead of glycine, at position 110 of the fusion protein, just preceding the cleavage site. Five of six virulent isolates, whose predicted motif was different from that usually found in virulent strains, also tested negative. However, the antibody did react with one isolate with the motif (112)KRQ(114). There was no apparent reactivity with any of the avirulent isolates tested. We conclude that this antibody may, in the future, be a useful aid for the pathotyping of NDV isolates.

A survey of the prevalence of stereotypy, self-injury and aggression in children and young adults with Cri du Chat syndrome.

The aim of the present study was to determine the prevalence and frequency of stereotypy, self-injurious behaviour (SIB), and aggression in children and adults with Cri du Chat syndrome (CCS), and to investigate the relationship between SIB, aggressive behaviour and stereotypy in these individuals. Sixty-six families of children and adults diagnosed with CCS completed the Behaviour Problems Inventory. Additional information relating to gender, chronological age, type of school/post-school occupation and medication was also included in the survey. Stereotyped behaviour was reported for 82% of subjects, more than half the sample displaying it on a daily basis. The occurrence percentage of 15 topographies of SIB suggested that head banging, hitting the head against body parts, self-biting and rumination are the most frequently occurring behaviours in CCS. Aggressive behaviour was reported for 88%, with a statistically significant negative correlation between age and the number of aggressive behaviours reported. The present findings suggest that specific types of stereotypy and SIB are observed frequently in CCS.

Growth study of cri du chat syndrome.

We compared the growth of children with cri du chat (5p-) syndrome with the 1990 UK growth curves. Most subjects had impaired growth, particularly of head circumference. The more emaciated the child the more pronounced the microcephaly, showing the need for growth and nutrition monitoring.

Widespread occurrence of Pneumocystis carinii in commercial rat colonies detected using targeted PCR and oral swabs.

The genus Pneumocystis contains a family of fungal organisms that infect a wide variety of mammalian species. Although it is a cause of pneumonia in immunocompromised hosts, recent evidence suggests that these organisms colonize nonimmunosuppressed hosts. Detection of cryptic colonization with Pneumocystis becomes important in animal studies when infection-free animals are necessary. Provocation by chronic immunosuppression, histology, and serology has been widely used to detect the presence of Pneumocystis in rat colonies, requiring lengthy time periods and/or postmortem tissue. We conducted a study to evaluate the use of PCR amplification of oral swabs for the antemortem detection of Pneumocystis in 12 rat groups from three commercial vendors. Sera were collected upon arrival, and the oral cavity was swabbed for PCR analysis. Ten of these groups of rats were then housed in pairs under barrier and immunosuppressed to provoke Pneumocystis growth. Once moribund, the rats were sacrificed, and the lungs were collected to evaluate the presence of Pneumocystis by PCR and microscopic enumeration. DNA was extracted from oral swabs and lung homogenates, and PCR was performed using primers targeting a region within the mitochondrial large-subunit rRNA of Pneumocystis carinii f. sp. carinii. Upon receipt, 64% of rats were positive for P. carinii f. sp. carinii-specific antibodies, while P. carinii f. sp. carinii DNA was amplified from 98% of oral swabs. Postmortem PCR analysis of individual lungs revealed P. carinii f. sp. carinii DNA in all rat lungs, illustrating widespread occurrence of Pneumocystis in commercial rat colonies. Thus, oral swab/PCR is a rapid, nonlethal, and sensitive method for the assessment of Pneumocystis exposure.

Rapid pathotyping of Newcastle disease virus (NDV) using fluorogenic probes in a PCR assay.

Hybridisation of PCR fragments with fluorogenic probes specific for pathotype allowed an estimation of pathogenicity of Newcastle disease virus (NDV) isolates using a modified TaqMan procedure. Six probes were used, designed to recognise nucleotide sequences in the fusion protein gene sequence corresponding to the precursor protein F0 cleavage site of both virulent and avirulent viruses. Forty-three of the 45 isolates tested, including 18 examined in a blind study were pathotyped successfully and rapidly, with close correlation between cleavage site nucleotide sequences, TaqMan results and intracerebral pathogenicity index (ICPI) values. One isolate, which could not be pathotyped by nucleotide sequencing, was shown using the TaqMan system to be a mixture of virulent and avirulent NDV. The results of this study suggest that using this modified TaqMan protocol, the likely virulence of most ND isolates can be determined rapidly and reproducibly.

Inhibitors of sterol biosynthesis and amphotericin B reduce the viability of pneumocystis carinii f. sp. carinii.

Pneumocystis carinii synthesizes sterols with a double bond at C-7 of the sterol nucleus and an alkyl group with one or two carbons at C-24 of the side chain. Also, some human-derived Pneumocystis carinii f. sp. hominis strains contain lanosterol derivatives with an alkyl group at C-24. These unique sterols have not been found in other pathogens of mammalian lungs. Thus, P. carinii may have important differences in its susceptibility to drugs known to block reactions in ergosterol biosynthesis in other fungi. In the present study, inhibitors of 3-hydroxy-3-methyglutaryl coenzyme A reductase, squalene synthase, squalene epoxidase, squalene epoxide-lanosterol cyclase, lanosterol demethylase, Delta(8) to Delta(7) isomerase, and S-adenosylmethionine:sterol methyltransferase were tested for their effects on P. carinii viability as determined by quantitation of cellular ATP levels in a population of organisms. Compounds within each category varied in inhibitory effect; the most effective included drugs targeted at squalene synthase, squalene epoxide-lanosterol cyclase, and Delta(8) to Delta(7) isomerase. Some drugs that are potent against ergosterol-synthesizing fungi had little effect against P. carinii, suggesting that substrates and/or enzymes in P. carinii sterol biosynthetic reactions are distinct. Amphotericin B is ineffective in clearing P. carinii infections at clinical doses; however, this drug apparently binds to sterols and causes permeability changes in P. carinii membranes, since it reduced cellular ATP levels in a dose-dependent fashion.

Integrative research review of risk behaviors among adolescents in rural, suburban, and urban areas.

PURPOSE: The purpose of this integrative review was to describe the state of the science regarding adolescent risk behaviors, with particular emphasis on comparisons among rural, urban, and suburban populations. METHOD: The review was done at two levels, moving from the major national survey studies which included data collected in the late 1980s up to 1993, to more focused topical areas including studies with data collection and publication between 1990 and 1996 within each identified category of adolescent health issues. A total of 137 published works across several disciplines were reviewed. Suggestions for clinical practice were drawn from the significant research findings. In addition, risk behaviors were compared to national baseline data and objectives. RESULTS: The level of research in this topic area was primarily descriptive. Currently, only a small portion of the national objectives for decreasing adolescent risk behaviors have been met. Successful intervention programs, although few in number, usually included not only topical education but also adolescent interaction with peers and support systems to raise awareness and change behaviors. CONCLUSIONS: The risk behaviors for the adolescent population as a whole have been well described. Education alone is not sufficient to change behaviors. Objective outcomes must be identified and health care providers need to use research findings in their practice with adolescents. It is time to intervene with developmentally and culturally appropriate strategies. There was a large gap in the literature regarding risk behaviors and protective factors for rural adolescents. The few studies that included subjects from rural settings indicated that the view that rural adolescents are engaged in fewer or less severe risk behaviors is misleading.

Antigenic and genetic characterisation of Newcastle disease viruses isolated from outbreaks in domestic fowl and turkeys in Great Britain during 1997.

Antigenic and genetic analyses of viruses from the 11 outbreaks of Newcastle disease in Great Britain, 12 of the outbreaks in Northern Ireland and the single outbreak in the Republic of Ireland which occurred in 1997, indicated that they were all essentially similar. In addition, the viruses from the British Isles were very similar to viruses isolated from three outbreaks in pheasants in Denmark between August and November 1996, from a goosander in Finland in September 1996, from an outbreak in chickens in Norway in February 1997, and from an outbreak in chickens in Sweden in November 1997. Viruses from outbreaks in other countries during 1995 to 1997 could be distinguished antigenically and/or genetically from the 1996 to 1997 Scandinavian/British Isles isolates, as could viruses responsible for two separate outbreaks in caged birds in quarantine premises in Great Britain in March 1997. Minor nucleotide differences in the 413-base region of the fusion gene and the 187-base region of the haemagglutinin-neuraminidase gene sequenced in this study allowed the 1996 to 1997 Scandinavian/British Isles isolates to be divided into groups. These groups broadly corresponded to the clusters of disease outbreaks, but suggested that the discrete outbreak in Scotland was probably the result of virus spread from Northern Ireland. Overall, the antigenic and genetic analyses of these viruses were consistent with the theory that the virus was introduced into the British Isles by migratory birds moving from north-east Europe. However, it was not possible to rule out other sources, such as the movement of pheasants from Denmark.

Experimental assessment of the pathogenicity of the Newcastle disease viruses from outbreaks in Great Britain in 1997 for chickens and turkeys, and the protection afforded by vaccination.

The Newcastle disease virus isolated from healthy turkeys in outbreak GB 97/6 was used to challenge 4-week-old turkeys and chickens, which were either not vaccinated or had received a single dose of Hitchner B1 live vaccine 14 days earlier, by one of the intramuscular, intranasal or contact routes. Similar experiments were done in 38-day-old turkeys and chickens using virus isolated from severely sick chickens in outbreak GB 97/1. All vaccinated chickens showed low but measurable immune responses 14 days after vaccination, but only three of the turkeys had detectable antibodies. No vaccinated turkey or chicken showed any clinical sign after challenge with either virus. The virus from healthy turkeys in outbreak GB 97/6 induced clinical signs in 12/30 unvaccinated turkeys after challenge and 7/30 died. In unvaccinated chickens, challenge with this virus produced clinical signs in 25/30 birds and 21/30 died. In challenge experiments with the virus from outbreak GB 97/1 in chickens, 3/30 unvaccinated turkeys showed clinical signs and all three subsequently died. In contrast, 30/30 unvaccinated chickens challenged with this virus showed clinical signs and died. Vaccination did not prevent infection and excretion of either challenge virus. However, when compared with unvaccinated birds, vaccination reduced significantly the length of time virus was excreted and the overall proportion of swabs that were positive.

Long-term results of radiologic placement of a central vein access device.

OBJECTIVE: We describe our long-term experience with radiologic implantation of the Peripheral Access System (PAS) Port venous access device. Technical efficacy and complications are documented and compared with surgical and radiologic series involving other long-term venous access devices. SUBJECTS AND METHODS: Fifty-two PAS-Port catheters were implanted in 51 patients during a 30-month period. All procedures took place in the angiography suite and were performed by interventional radiologists with imaging guidance. Patients were followed up through the oncology clinic or the clinic that originally referred the patient. The durability of the catheter was evaluated, and complications were recorded during the study period. RESULTS: Fifty-two ports have been indwelling for a total of 18,357 patient-days. The mean time of implantation was 372 days, with a range of 30-825 days. Technical success in implanting the device was 100%. Device-related sepsis occurred in one patient (2%), superficial thrombophlebitis in one patient (2%), skin site dehiscence in one patient (2%), and deep vein thrombosis in one patient (2%). No instances of catheter occlusion occurred, and all catheters retained the ability to aspirate blood throughout their use. The overall complication rate was 8% (0.22/1000 patient days). CONCLUSION: Radiologic placement of this device is safe and effective. It offers many patients a superior alternative to surgically implanted chest wall ports. Complications are fewer, and chances for technical success are greater. In circumstances where cosmesis is deemed highly important, the PAS-Port device may be preferable to tunneled venous access catheters.

Antigenic and phylogenetic studies on a variant Newcastle disease virus using anti-fusion protein monoclonal antibodies and partial sequencing of the fusion protein gene.

A virulent Newcastle disease virus (NDV) isolate, 34/90, reported to show considerable antigenic diversity from more classical strains of NDV, including vaccine strains, was evaluated phylogenetically and for the presence of neutralizing epitopes on the fusion protein. Comparison of a 309 nucleotide sequence of the fusion protein gene of 34/90 with other viruses confirmed the diversity of this virus, placing it in a discrete fifth genetic lineage with an avirulent virus isolated from waterfowl and genetically quite distant from other strains and isolates. The virus-neutralizing mAbs used in the present study were directed against at least seven distinct epitopes on the fusion protein. Of these seven, five are shared by 34/90 and the live vaccine virus Hitchner B1 and these plus an additional epitope are shared by 34/90 and strain Ulster 2C, which is used in inactivated vaccines. Two potential distinct epitopes were also shared by these three viruses. The results suggest that despite the detected antigenic and genetic variation of 34/90, it is unlikely that mutants which fail to be neutralized by antibodies induced by conventional vaccines would arise readily.

Antigenic diversity and similarities detected in avian paramyxovirus type 1 (Newcastle disease virus) isolates using monoclonal antibodies.

Newcastle disease (ND) virus (APMV-1) isolates submitted to the International Reference Laboratory for ND were characterised antigenically by their ability to cause binding of mouse monoclonal antibodies (mAbs) to cell cultures infected with the isolate. Since the availability of the mAbs 1526 viruses have been examined using a panel of nine mAbs and 818 with an extended panel of 26 mAbs. Using the nine mAb panel a total of 14 different patterns was seen and viruses grouped by the same pattern showed relationships with each other which were either biological, temporal or geographical or more than one of these. There was a marked tendency of viruses placed in the same group to show similar virulence for chickens. Extension of the panel to 26 mAbs produced 39 distinct patterns, although some of these were seen with only a single virus. Again, viruses inducing similar binding patterns shared similar properties and some binding patterns were specific for viruses causing discrete epizootics. Cluster analysis of the mAb binding patterns did not produce concise, discrete groupings, but did emphasise some relationships between virus properties and antigenicity. Examples of the usefulness of this approach were the ability to link two important outbreaks to the contamination of stored food by infected feral pigeons, and the demonstration of two separate viruses responsible for outbreaks in countries of the European Union during 1991 to 1994 thus preventing erroneous epizootiological tracing.