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.

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 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.

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.

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.

Pathogenicity and phylogenetic evaluation of the variant Newcastle disease viruses termed "pigeon PMV-1 viruses" based on the nucleotide sequence of the fusion protein gene.

The nucleotide sequences of the entire F genes of two isolates of the pigeon PMV-1 (PPMV-1) variant of Newcastle disease virus (NDV) were determined using RTPCR. The deduced amino acid sequences of the F0 protein showed four differences between isolate 760/83 which had been passaged 4 times in chickens and gave an intravenous pathogenicity index in chickens (IVPI) of 2.01 and isolate 1168/84 which had received six passages in chickens and had an IVPI of 0.00. The F genes of virus from two passage levels of isolate 1447/84, 0 with IVPI value 0.00 and six with IVPI value 0.58, were partially sequenced to cover the areas of variation between 760/83 and 1168/84. The two passage levels of 1447/84 showed identical sequences in these areas which in turn were identical of those of 760/83. It was concluded that the recorded differences in intravenous pathogenicity were unlikely to be associated with differences in the primary structure of the F0 protein. Phylogenetic comparisons of the F gene sequences of the two PPMV-1 viruses with those published for other NDV strains and isolates showed that the PPMV-1 viruses formed a new fourth lineage but were closely related to strain Warwick with which they presumably shared a common origin.

Evaluation of the molecular basis of pathogenicity of the variant Newcastle disease viruses termed "pigeon PMV-1 viruses".

The amino acid sequence at the F2/F1 cleavage site was determined for 15 strains of the so-called pigeon PMV-1 (PPMV-1) variant of Newcastle disease virus (NDV) which showed close antigenic identity, determined by their reactions with a panel of 28 monoclonal antibodies, but considerable variation in their pathogenicity for chickens. Thirteen of the isolates possessed the motif 112G-R-Q-K-R-F117. This motif was seen for one virus which had initially low pathogenicity and remained unaltered when virulence of the virus for chickens was increased by bird to bird passage. The two other viruses had the sequence 112R-R-Q-K-R-F117 at the cleavage site which is more typical of virulent viruses, however, pathogenicity index tests indicated that these isolates were of moderate and low pathogenicity. The nucleotide sequence coding for the HN/HN0 extension region was determined for two of the PPMV-1 isolates. In both cases a stop codon was present indicating that the product for these viruses would be HN571. We conclude that the wide variation in pathogenicity of the variant PPMV-1 for chickens is not related to variation in the amino acid motif at the F2/F1 cleavage site nor due to production of HN0 which may also influence pathogenicity. The high virulence of some of the viruses examined confirms that a double pair of basic amino acids in the region of the F2/F1 cleavage site is not necessary for the full expression of virulence.

Deduced amino acid sequences at the fusion protein cleavage site of Newcastle disease viruses showing variation in antigenicity and pathogenicity.

The amino acid sequence at the F2/F1 cleavage site of the F0 fusion protein of 17 strains of Newcastle disease virus (NDV) was deduced from sequencing a 32 nucleotide area of the genome by reverse transcription and polymerase chain reaction (PCR) techniques. With the addition of sequences at the same area previously published for 9 other viruses comparisons were made of a total of 26 NDV strains and isolates (11 of low virulence, 15 of high virulence or mesogenic) covering ten antigenic groups determined by reactions with monoclonal antibodies. All the virulent viruses and the mesogenic strain Komarov showed the amino acid sequence 112R/K-R-Q-K/R-R116 for the C-terminus of the F2 protein and phenylalanine (F) at the N-terminus of the F1 protein, residue 117. The mesogenic isolate of the antigenic variant NDV responsible for the recent panzootic in racing pigeons, often termed "pigeon paramyxovirus type 1", examined in this study had the sequence 112G-R-Q-K-R-F117. The deduced amino acid sequence in the corresponding region of all viruses of low virulence was 112G/E-K/R-Q-G/E-R-L117. The virulent virus, PMV-1/chicken/Ireland/34/90 (34/90), which had a close antigenic relationship to a group of avirulent viruses, three of which were examined in the present study as representatives of the monoclonal antibody group H, showed between 4-6 nucleotide differences from these viruses in the 32 nucleotide region studied. These resulted in differences in the deduced amino acid sequence at residue 112 E-->K, 115 E-->K and 117-->F, giving 34/90 a typical virulent virus motif at the cleavage site. Despite the extremely small portion of the genome studied there were several areas which appeared characteristic for 34/90 and the three group H viruses of low virulence, which suggests that they may have arisen from the same gene pool.

The role of bactericidal/permeability-increasing protein as a natural inhibitor of bacterial endotoxin.

Systemic release of endotoxin (LPS) after Gram-negative infection initiates a cascade of host cytokines that are thought to be the direct cause of shock, multisystem organ failure, and death. Endogenous LPS-binding proteins may play a role in regulating LPS toxicity in vivo. The human neutrophil granule protein bactericidal/permeability-increasing protein (BPI) shares sequence homology and immunocrossreactivity with an acute phase lipopolysaccharide binding protein (LBP) which has been shown to bind to LPS and accelerate LPS activation of neutrophils and macrophages. Although structurally similar, LBP and BPI are apparently functionally antagonistic. We previously showed that BPI inhibits LPS-mediated neutrophil activation in vitro. Here we demonstrate that BPI binds to LPS near the lipid A domain, and formation of the LPS-BPI complex abrogates detrimental host responses to LPS. For example, BPI blocks LPS-stimulated TNF release in vitro and in vivo, and LPS complexed to BPI is not pyrogenic in rabbits. Results demonstrating that BPI is released by stimulated human neutrophils further support the idea that BPI functions extracellularly in vivo to neutralize endotoxin. Taken together, these data argue that BPI neutralizes the toxic effects of LPS in vivo, and that BPI may represent a new therapeutic approach to the treatment of endotoxic shock.

Characterisation of an antigenically unusual virus responsible for two outbreaks of Newcastle disease in the Republic of Ireland in 1990.

Antigenic characterisation of two highly virulent virus isolates from outbreaks of Newcastle disease on two closely connected farms in County Monaghan, Republic of Ireland, in 1990 showed the viruses to be indistinguishable but unlike other Newcastle disease viruses so far tested. However, they appeared to be antigenically closest to avirulent viruses isolated from waterfowl from several countries and from chickens in Northern Ireland in 1986. Despite the antigenic differences, chickens vaccinated with a live commercial Hitchner B1 vaccine were protected against intramuscular challenge with one of the virulent isolates.

Evaluation of relationships between avian paramyxoviruses isolated from birds of the family Columbidae.

The prototype virus for the PMV-7 serotype of avian paramyxoviruses, PMV-7 dove Tennessee/4/75 (Tn 4) and five other isolates obtained from birds of the Columbidae family, which had been shown to be distinct from PMV-1 serotype, were tested for antigenic relationships between themselves and to other avian paramyxoviruses. By serological tests and analysis of structural polypeptides the viruses appeared to be distinct from other avian paramyxoviruses. One isolate appeared to be very closely related to Tn/4. Three other isolates showed only minor relationships to these two but were very closely related to each other. However, the sixth virus, pigeon Japan/Otaru/76, showed high levels of homology in haemagglutination inhibition tests and at least one line of identity in immunodoublediffusion tests with all five of the other isolates.

Viruses and virus-like particles detected in samples from diseased game birds in Great Britain during 1988.

During 1988, 108 samples were received from game birds (78 from pheasants, 28 from partridges and two from quail) for virus isolation or detection; 89 being received during the June to August rearing period. The most common clinical signs resulting in the submissions were death, scour and stunting. Virus or virus-like particles were detected in 51 cases, 43 as a result of direct electron microscopy of gut contents, seven by agar gel precipitin test for the presence of Marble spleen disease antigen and one by isolation, of a rotavirus. Particles observed by electron microscopy were: rotavirus - 15, adenovirus - 1, reovirus - 1, enterovirus - 1, 'fimbriated' virus-like particles - 10, rod-shaped virus-like particles - 19, On three occasions more than one type of particle was seen in the same sample.

Pseudomonas hyperimmune globulin passive immunotherapy for pulmonary exacerbations in cystic fibrosis.

We studied the effect of an intravenously administered gamma globulin [Ps-ivIG] enriched fivefold over conventional ivIG for Pseudomonas aeruginosa lipopolysaccharide [PA LPS] antibodies on ten patients with cystic fibrosis [CF] aged 19-32 years during hospitalization for pulmonary deterioration. All were colonized with greater than or equal to 1 PA phenotype resistant to all antibiotics at the time of admission and they received 500 mg/kg Ps-ivIG intravenously as a single dose in addition to conventional treatment, including antibiotics and chest physiotherapy. No adverse effects occurred. Circulating immune complexes and complement levels remained unchanged from baseline. Serum levels of anti-PA LPS IgG, as measured by ELISA for eight PA LPS immunotypes, increased to 244 +/- 65% (mean +/- SE) of baseline levels 1 hour post-infusion (P less than 0.01), remained significantly elevated during a mean hospital stay of 17 days, and returned to near baseline by follow-up 4 weeks after hospital discharge. Plasma half-life and clearance values were similar to those of other subjects receiving conventional ivIG. Sputum PA density declined from 3.0 to 1.2 x 10(8) cfu/mL 1 week post-infusion (P approximately equal to 0.05), and returned to baseline at follow-up. Serum anti-PA opsonic activity increased after infusion (P less than 0.01), but returned to baseline by 72 hours. Clinical scores improved from admission to discharge (P less than 0.005) without decline at follow-up. Forced vital capacity [FVC] and forced expiratory volume in one second [FEV1] increased from admission to discharge (P less than 0.01 and P less than 0.05, respectively) without decline at follow-up. Using autologous historical control data, standard hospital therapy without Ps-ivIG resulted in no improvement in FVC or FEV1, and a subsequent decline in these parameters (P less than 0.05 for each) during a similar follow-up period. This occurred despite the fact that half the patients did not have antibiotic-resistant PA on the control admission. We conclude that Ps-ivIG is a safe adjunctive therapy for pulmonary exacerbations in moderately ill cystic fibrosis patients colonized with resistant PA, and may be associated with both greater and more prolonged improvement in pulmonary function than standard therapy alone.

Location of neutralizing epitopes on the fusion protein of Newcastle disease virus strain Beaudette C.

A panel of eight neutralizing monoclonal antibodies (MAbs) against the fusion (F) protein of Newcastle disease virus (NDV) has been shown to locate a major antigenic site on the basis of competitive binding assay and additivity index studies. Five epitopes (A1 to A5) have been located within this site on the F protein of the Beaudette C strain of NDV on the basis of cross-resistance plaque assays of MAb-resistant mutants raised against these MAbs. Epitopes A1, A4 and A5 are distinct; epitope A2 partially overlaps epitope A3. Nucleotide sequence analysis of the F genes of MAb-resistant mutants showed that each predicted single amino acid substitutions ranging from amino acid residues 157 to 171 for epitope A4 and at residues 72, 78, 79 and 343 for epitopes A1, A2, A3 and A5 respectively. These locations indicate that both the F1 and F2 fragments are involved in the formation of a single antigenic site and suggest the involvement of extensive protein folding in the active form of this F protein.

Antigenic relationships of three turkey rhinotracheitis viruses.

Three turkey rhinotracheitis isolates obtained from different laboratories were compared by electron microscopy, polypeptide analysis, double immunodiffusion and cross neutralisation tests. The results obtained showed that the three viruses were morphologically and antigenically similar. It is suggested that turkey rhinotracheitis viruses belong to the Pneumovirus genus.

Evaluation of mouse monoclonal antibodies raised against an isolate of the variant avian paramyxovirus type 1 responsible for the current panzootic in pigeons.

Nine monoclonal antibodies raised against "pigeon variant" avian paramyxovirus type 1 isolate pigeon/England/617/83 were tested for their ability to react with "classical" and other "pigeon variant" isolates. Two of the monoclonal antibodies appeared to be specific for 617/83 reacting with no other virus. The remaining seven monoclonal antibodies bound to cells infected with all other "pigeon" isolates in indirect immunoperoxidase (IIP) tests but four distinct groups of other PMV-1 viruses were formed on the basis of the binding patterns. One of the monoclonal antibodies 161/617 caused haemagglutination inhibition (HI) of all the "pigeon" isolates tested but none of the other PMV-1 viruses and these results reflected the IIP results with this monoclonal antibody. 161/617 was also shown to inhibit viruses of the avian paramyxovirus type 3 serogroup in HI tests. This reaction and the ability to bind to infected cells in IIP tests appeared to be restricted to PMV-3 viruses isolated from exotic birds and did not occur with viruses of ostensively the same serotype from turkeys.

Characterization of paramyxoviruses isolated from penguins in Antarctica and sub-Antarctica during 1976-1979.

Nine paramyxovirus isolates obtained from penguins were tested for antigenic relationships amongst themselves and to other avian paramyxoviruses. One of the isolates was shown to be a lentogenic Newcastle disease virus (NDV), i.e., of PMV-1 serotype. By serological tests and analysis of structural polypeptides the other penguin isolates could be placed into three groups. No relationship with other avian paramyxoviruses could be determined except that six of the penguin viruses, representing two of the groups, showed reaction with a monoclonal antibody raised against NDV Ulster 2C and three of the isolates, representing one of the penguin groups, also reacted with another PMV-1 directed monoclonal antibody.

Characterization of a virus associated with turkey rhinotracheitis.

A virus associated with turkey rhinotracheitis was purified and its morphology and structural polypeptides were compared with those of the bovine, human and murine members of the genus Pneumovirus. The isolate possessed surface projections 13 to 14 nm in length and a helical nucleocapsid 14 nm in diameter with a pitch of 7 nm. Approximately seven presumed viral polypeptides were observed. Their apparent molecular weights were 200 x 10(3) (200K), 84K, 54K, 42K, 37K, 31K and 14K; two of these, the 84K and 54K polypeptides, were glycosylated. The virus was shown to possess many features that were similar to established pneumoviruses and can therefore be regarded as a possible member of this genus.

Routine virus isolation or detection in the diagnosis of diseases in birds.

Between August 1986 and July 1987, 373 submissions for laboratory diagnosis of suspected virus diseases of birds were received. In 116 cases (31%) viruses were isolated or detected, consisting of 25 reo-viruses, 17 rotaviruses, 14 adenoviruses plus demonstration of haemorrhagic enteritis virus antigen in the spleens of 15 birds, eight pox-viruses, 19 duck enteritis herpesviruses and six other avian herpes-viruses, four duck hepatitis viruses, six other enterovirus-like isolates, two duck astroviruses and two avian infectious bronchitis viruses.