Haemophilus influenzae genome evolution during persistence in the human airways in chronic obstructive pulmonary disease.

Nontypeable Haemophilus influenzae (NTHi) exclusively colonize and infect humans and are critical to the pathogenesis of chronic obstructive pulmonary disease (COPD). In vitro and animal models do not accurately capture the complex environments encountered by NTHi during human infection. We conducted whole-genome sequencing of 269 longitudinally collected cleared and persistent NTHi from a 15-y prospective study of adults with COPD. Genome sequences were used to elucidate the phylogeny of NTHi isolates, identify genomic changes that occur with persistence in the human airways, and evaluate the effect of selective pressure on 12 candidate vaccine antigens. Strains persisted in individuals with COPD for as long as 1,422 d. Slipped-strand mispairing, mediated by changes in simple sequence repeats in multiple genes during persistence, regulates expression of critical virulence functions, including adherence, nutrient uptake, and modification of surface molecules, and is a major mechanism for survival in the hostile environment of the human airways. A subset of strains underwent a large 400-kb inversion during persistence. NTHi does not undergo significant gene gain or loss during persistence, in contrast to other persistent respiratory tract pathogens. Amino acid sequence changes occurred in 8 of 12 candidate vaccine antigens during persistence, an observation with important implications for vaccine development. These results indicate that NTHi alters its genome during persistence by regulation of critical virulence functions primarily by slipped-strand mispairing, advancing our understanding of how a bacterial pathogen that plays a critical role in COPD adapts to survival in the human respiratory tract.

Gestation as mothering.

Some commentators maintain that gestational surrogates are not 'mothers' in a way capable of grounding a claim to motherhood. These commentators find that the practices that constitute motherhood do not extend to gestational surrogates. We argue that gestational surrogates should be construed as mothers of the children they bear, even if they fully intend to surrender those children at birth to the care of others. These women stand in a certain relationship to the expected children: they live in changed moral circumstances by reason of their pregnancy, and they engage in the practices said to define motherhood in the post-birth context. By contrast, ovum donors and embryo donors are not similarly 'mothers' because they do not find themselves involved in these circumstances. Not all women involved in three-parent in vitro fertilization qualify as mothers either. Given this analysis of mothering, we note that transmen who gestate children are engaged in mothering activity even if they otherwise function as a father to those children. By itself, this defence of the maternity of gestational surrogates does not confer moral title to the children they bear; gestation would not by itself override the contractual arrangements gestational surrogates have made regarding the disposition of their children. This interpretation of gestational surrogates as mothers does, however, undercut cultural understandings of these women as mere 'vessels', devoid of entitlement to respect as persons and parents. We also consider the meaning of mothering for 'brain-dead' women kept alive to give birth and for the prospect of extracorporeal gestation.

The Laminin Interactome: A Multifactorial Laminin-Binding Strategy by Nontypeable Haemophilus influenzae for Effective Adherence and Colonization.

Laminin is a well-defined component of the airway basement membrane (BM). Efficient binding of laminin via multiple interactions is important for nontypeable Haemophilus influenzae (NTHi) colonization in the airway mucosa. In this study, we identified elongation factor thermo-unstable (EF-Tu), l-lactate dehydrogenase (LDH), protein D (PD), and peptidoglycan-associated lipoprotein P6 as novel laminin-binding proteins (Lbps) of NTHi. In parallel with other well-studied Lbps (protein 4 [P4], protein E [PE], protein F [PF], and Haemophilus adhesion and penetration protein [Hap]), EF-Tu, LDH, PD, and P6 exhibited interactions with laminin, and mediated NTHi laminin-dependent adherence to pulmonary epithelial cell lines. More importantly, the NTHi laminin interactome consisting of the well-studied and novel Lbps recognized laminin LG domains from the subunit α chains of laminin-111 and -332, the latter isoform of which is the main laminin in the airway BM. The NTHi interactome mainly targeted multiple heparin-binding domains of laminin. In conclusion, the NTHi interactome exhibited a high plasticity of interactions with different laminin isoforms via multiple heparin-binding sites.

Persistence of Moraxella catarrhalis in Chronic Obstructive Pulmonary Disease and Regulation of the Hag/MID Adhesin.

BACKGROUND: Persistence of bacterial pathogens in the airways has profound consequences on the course and pathogenesis of chronic obstructive pulmonary disease (COPD). Patients with COPD continuously acquire and clear strains of Moraxella catarrhalis, a major pathogen in COPD. Some strains are cleared quickly and some persist for months to years. The mechanism of the variability in duration of persistence is unknown. METHODS: Guided by genome sequences of selected strains, we studied the expression of Hag/MID, hag/mid gene sequences, adherence to human cells, and autoaggregation in longitudinally collected strains of M. catarrhalis from adults with COPD. RESULTS: Twenty-eight of 30 cleared strains of M. catarrhalis expressed Hag/MID whereas 17 of 30 persistent strains expressed Hag/MID upon acquisition by patients. All persistent strains ceased expression of Hag/MID during persistence. Expression of Hag/MID in human airways was regulated by slipped-strand mispairing. Virulence-associated phenotypes (adherence to human respiratory epithelial cells and autoaggregation) paralleled Hag/MID expression in airway isolates. CONCLUSIONS: Most strains of M. catarrhalis express Hag/MID upon acquisition by adults with COPD and all persistent strains shut off expression during persistence. These observations suggest that Hag/MID is important for initial colonization by M. catarrhalis and that cessation of expression facilitates persistence in COPD airways.

ReVac: a reverse vaccinology computational pipeline for prioritization of prokaryotic protein vaccine candidates.

BACKGROUND: Reverse vaccinology accelerates the discovery of potential vaccine candidates (PVCs) prior to experimental validation. Current programs typically use one bacterial proteome to identify PVCs through a filtering architecture using feature prediction programs or a machine learning approach. Filtering approaches may eliminate potential antigens based on limitations in the accuracy of prediction tools used. Machine learning approaches are heavily dependent on the selection of training datasets with experimentally validated antigens (positive control) and non-protective-antigens (negative control). The use of one or few bacterial proteomes does not assess PVC conservation among strains, an important feature of vaccine antigens. RESULTS: We present ReVac, which implements both a panoply of feature prediction programs without filtering out proteins, and scoring of candidates based on predictions made on curated positive and negative control PVCs datasets. ReVac surveys several genomes assessing protein conservation, as well as DNA and protein repeats, which may result in variable expression of PVCs. ReVac's orthologous clustering of conserved genes, identifies core and dispensable genome components. This is useful for determining the degree of conservation of PVCs among the population of isolates for a given pathogen. Potential vaccine candidates are then prioritized based on conservation and overall feature-based scoring. We present the application of ReVac, applied to 69 Moraxella catarrhalis and 270 non-typeable Haemophilus influenzae genomes, prioritizing 64 and 29 proteins as PVCs, respectively. CONCLUSION: ReVac's use of a scoring scheme ranks PVCs for subsequent experimental testing. It employs a redundancy-based approach in its predictions of features using several prediction tools. The protein's features are collated, and each protein is ranked based on the scoring scheme. Multi-genome analyses performed in ReVac allow for a comprehensive overview of PVCs from a pan-genome perspective, as an essential pre-requisite for any bacterial subunit vaccine design. ReVac prioritized PVCs of two human respiratory pathogens, identifying both novel and previously validated PVCs.

Biogenetic ties and parent-child relationships: The misplaced critique.

According to an almost axiomatic standard in bioethics, moral commitment should ground parents' relationship with their children, rather than biogenetic relatedness. This standard has been used lately to express skepticism about extending existing assisted reproductive treatments (ARTs) to same-sex couples and to research into novel fertility interventions for those couples, but this skepticism is misplaced on several grounds. As a matter of access and equity, same-sex couples seem presumptively entitled to genetic relatedness to their children as far as possible both in regard to existing ARTs and to novel ARTs under investigation. For those worried about the effects of trying to secure biogenetic relatedness for same-sex couples, it may be noted that same-sex couples will only ever be a fraction of the parents implicated in propping up "biologism," as the expectation of biogenetic relatedness it is sometimes called. The cultural force of biologism would survive almost intact even if no same-sex couples were ever to have genetically related children. It is therefore hard to see why same-sex couples should forfeit aspirations to biogenetic relationships with their children or enjoy less subsidy for ARTs than the subsidy given to different-sex couples. As matter of moral consistency, the full implications of the biologism critique have yet to be evaluated relative to different-sex couples.

Adolescents and Body Modification for Gender Identity Expression.

As a matter of ethics and law, adults enjoy wide berth in securing hormonal and surgical interventions to align their bodies with their desired gender appearance. In contrast, the exercise of choice by minors is more constrained, because they can be less well situated to grasp the nature and consequences of interventions having life-long effects. Even so, some minors hope for body modifications prior to adulthood. Starting very young, some minors may assert atypical gender identity: those with female-typical bodies assert a male identity and those with male-typical bodies assert a female identity. This assertion of identity is atypical only in a descriptive sense, because it is uncharacteristic, not because it is normatively unacceptable. Not all minors persist in their atypical gender identities, but some do. For those who do, it is desirable to minimize unwanted secondary sex characteristics and to maximize desired secondary sex characteristics. I outline here a theory of respect for decisions by minors in regard to hormonal and surgical interventions that help align their bodies with their gender identity. Of particular ethical interest here are body modifications for fertility preservation since certain interventions in the body can leave people unable to have genetically related children. In general, I will show that the degree of respect owed to minors in regard to body modifications for gender identity expression should be scaled according to their decision-making capacities, in the context of robust practices of informed consent.

Mapping Protective Regions on a Three-Dimensional Model of the Moraxella catarrhalis Vaccine Antigen Oligopeptide Permease A.

A vaccine against Moraxella catarrhalis would reduce tremendous morbidity, mortality, and financial burden by preventing otitis media in children and exacerbations of chronic obstructive pulmonary disease (COPD) in adults. Oligopeptide permease A (OppA) is a candidate vaccine antigen that is (i) a nutritional virulence factor expressed on the bacterial cell surface during infection, (ii) widely conserved among strains, (iii) highly immunogenic, and (iv) a protective antigen based on its capacity to induce protective responses in immunized animals. In the present study, we show that the antibodies to OppA following vaccination mediate accelerated clearance in animals after pulmonary challenge. To identify regions of OppA that bind protective antibodies, truncated constructs of OppA were engineered and studied to map regions of OppA with surface-accessible epitopes that bind high-avidity antibodies following vaccination. Protective epitopes were located in the N and C termini of the protein. Immunization of mice with constructs corresponding to these regions (T5 and T8) induced protective responses. Studies of overlapping peptide libraries of constructs T5 and T8 with OppA immune serum identified two discrete regions on each construct. These potentially protective regions were mapped on a three-dimensional computational model of OppA, where regions with solvent-accessible amino acids were identified as three potentially protective epitopes. In all, these studies revealed two regions with three specific epitopes in OppA that induce potentially protective antibody responses following vaccination. Detection of antibodies to these regions could serve to guide vaccine formulation and as a diagnostic tool for monitoring development of protective responses during clinical trials.

Changes in IgA Protease Expression Are Conferred by Changes in Genomes during Persistent Infection by Nontypeable Haemophilus influenzae in Chronic Obstructive Pulmonary Disease.

Nontypeable Haemophilus influenzae (NTHi) is an exclusively human pathobiont that plays a critical role in the course and pathogenesis of chronic obstructive pulmonary disease (COPD). NTHi causes acute exacerbations of COPD and also causes persistent infection of the lower airways. NTHi expresses four IgA protease variants (A1, A2, B1, and B2) that play different roles in virulence. Expression of IgA proteases varies among NTHi strains, but little is known about the frequency and mechanisms by which NTHi modulates IgA protease expression during infection in COPD. To assess expression of IgA protease during natural infection in COPD, we studied IgA protease expression by 101 persistent strains (median duration of persistence, 161 days; range, 2 to 1,422 days) collected longitudinally from patients enrolled in a 20-year study of COPD upon initial acquisition and immediately before clearance from the host. Upon acquisition, 89 (88%) expressed IgA protease. A total of 16 of 101 (16%) strains of NTHi altered expression of IgA protease during persistence. Indels and slipped-strand mispairing of mononucleotide repeats conferred changes in expression of igaA1, igaA2, and igaB1 Strains with igaB2 underwent frequent changes in expression of IgA protease B2 during persistence, mediated by slipped-strand mispairing of a 7-nucleotide repeat, TCAAAAT, within the open reading frame of igaB2 We conclude that changes in iga gene sequences result in changes in expression of IgA proteases by NTHi during persistent infection in the respiratory tract of patients with COPD.

Azithromycin Pharmacodynamics against Persistent Haemophilus influenzae in Chronic Obstructive Pulmonary Disease.

The pharmacodynamic profile of azithromycin against persistent strains of nontypeable Haemophilus influenzae (NTHi) from chronic obstructive pulmonary disease (COPD) patients was characterized. Azithromycin displayed differential concentration-dependent activities (R2 ≥ 0.988); the pharmacodynamic response was attenuated when we compared the "first" and "last" strains of NTHi that persisted in the airways of the same patient for 819 days (the 50% effective concentration [EC50] increased more than 50 times [0.0821 mg/liter versus 4.23 mg/liter]). In the hollow-fiber infection model, NTHi viability was maintained throughout simulated azithromycin (Zithromax) Z-Pak regimens over 10 days.

Lower Airway Bacterial Colonization Patterns and Species-Specific Interactions in Chronic Obstructive Pulmonary Disease.

Little is known about interactions between nontypeable Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa in the lower respiratory tract in chronic obstructive pulmonary disease (COPD) patients. We characterized colonization by these four bacterial species, determined species-specific interactions, and estimated the effects of host factors on bacterial colonization among COPD patients. We conducted a prospective cohort study in veterans with COPD that involved monthly clinical assessment and sputum cultures with an average duration of follow-up of 4.5 years. Cultures were used for bacterial identification. We analyzed bacterial interactions using generalized linear mixed models after controlling for clinical and demographic variables. The outcomes of interest were the relationships between bacteria based on clinical status (stable or exacerbation). One hundred eighty-one participants completed a total of 8,843 clinic visits, 30.8% of which had at least one of the four bacteria isolated. H. influenzae was the most common bacterium isolated (14.4%), followed by P. aeruginosa (8.1%). In adjusted models, S. pneumoniae colonization was positively associated with H. influenzae colonization (odds ratio [OR], 2.79; 95% confidence interval [CI], 2.03 to 3.73). We identified negative associations between P. aeruginosa and H. influenzae (OR, 0.15; 95% CI, 0.10 to 0.22) and P. aeruginosa and M. catarrhalis (OR, 0.51; 95% CI, 0.35 to 0.75). Associations were similar during stable and exacerbation visits. Recent antimicrobial therapy was associated with a lower prevalence of S. pneumoniae, H. influenzae, and M. catarrhalis, but not P. aeruginosa Our findings support the presence of specific interspecies interactions between common bacteria in the lower respiratory tracts of COPD patients. Further work is necessary to elucidate the mechanisms of these complex interactions that shift bacterial species.

Differential distribution of IgA-protease genotypes in mucosal and invasive isolates of Haemophilus influenzae in Sweden.

BACKGROUND: Several different IgA-proteases exist in Haemophilus influenzae. The variants have been suggested to play differential roles in pathogenesis, but there is limited information on their distribution in clinical isolates. The objective of this study was to investigate the distribution of IgA-protease genotypes in H. influenzae and assess the association between IgA-protease genotype and type of clinical infection. METHODS: We performed PCR-screening of the IgA-protease gene variants in two cohorts of clinical H. influenzae. The first cohort consisted of 177 isolates from individuals with respiratory tract infection in January 2010, 2011 and 2012. Information on age, gender and clinical infection was available in this cohort. The second cohort comprised 53 isolates, including NTHi from bloodstream, cerebrospinal fluid (CSF) and urogenital origin as well as encapsulated isolates respresenting all capsule types. We assessed associations between IgA protease genotype and clinical predictors using basic statistical tests of association as well as regression analysis. RESULTS: The igaB gene was found in 46% of isolates in the respiratory tract cohort, and no evident trend could be seen during the study years. However, the igaB gene was significantly less common among invasive isolates (19%), p = 0.003 (Fischer's exact test), even when encapsulated isolates were excluded (21%), p = 0.012. A significantly negative association between bacteraemia and igaB genotype remained after adjusting for covariates. We did not identify a significant association between IgA-protease gene variants and type of respiratory tract infection, but isolates with an igaA2 genotype were overrepresented in pre-school children. CONCLUSIONS: The distribution of IgA-protease gene variants in Swedish H. influenzae highlighted the widespread abundance of the igaB in isolates from cases of respiratory tract infection, but the igaB gene variant was significantly less common in invasive (bloodstream and CSF) isolates of H. influenzae compared with respiratory tract isolates.

Pathways to genetic parenthood for same-sex couples.

Researchers are pursuing various ways to synthesise human male and female gametes, which would be useful for people facing infertility. Some people are unable to conceive children with their partner because one of them is infertile in the sense of having an anatomical or physiological deficit. Other people-in same sex couples-may not be individually infertile but situationally infertile in relation to one another. Segers et al have described a pathway towards synthetic gametes that would rely on embryonic stem cells, rather than somatic cells. This pathway would be advantageous, they say, for same-sex couples even though it would not offer those couples 50%-50% shared genetics in their children but only 50%-25%. It is unclear, however, why this approach should be preferred morally speaking since it represents a falling off from the kind of shared genetics in children that are functionally a gold standard in parents' expectations generally. Despite raising concerns about whether genetic relatedness is necessary or sufficient as a condition of parental interest in children, Segers et al cede the sociocultural importance of that standard. If so, same-sex couples seem entitled to press a case for some measure of research priority that would offer the same level of access to that social good as everyone else.

So not mothers: responsibility for surrogate orphans.

The law ordinarily recognises the woman who gives birth as the mother of a child, but in certain jurisdictions, it will recognise the commissioning couple as the legal parents of a child born to a commercial surrogate. Some commissioning parents have, however, effectively abandoned the children they commission, and in such cases, commercial surrogates may find themselves facing unexpected maternal responsibility for children they had fully intended to give up. Any assumption that commercial surrogates ought to assume maternal responsibility for abandoned children runs contrary to the moral suppositions that typically govern contract surrogacy, in particular, assumptions that gestational carriers are not 'mothers' in any morally significant sense. In general, commercial gestational surrogates are almost entirely conceptualised as 'vessels'. In a moral sense, it is deeply inconsistent to expect commercial surrogates to assume maternal responsibility simply because commissioning parents abandon children for one reason or another. We identify several instances of child abandonment and discuss their implications with regard to the moral conceptualisation of commercial gestational surrogates. We conclude that if gestational surrogates are to remain conceptualised as mere vessels, they should not be expected to assume responsibility for children abandoned by commissioning parents, not even the limited responsibility of giving them up for adoption or surrendering them to the state.

Bioethics, children, and the environment.

Queer perspectives have typically emerged from sexual minorities as a way of repudiating flawed views of sexuality, mischaracterized relationships, and objectionable social treatment of people with atypical sexuality or gender expression. In this vein, one commentator offers a queer critique of the conceptualization of children in regard to their value for people's identities and relationships. According to this account, children are morally problematic given the values that make them desirable, their displacement of other beings and things entitled to moral protection, not to mention the damaging environmental effects that follow in the wake of population growth. Objectionable views of children are said even to have colonized the view of lesbian, gay, bisexual, and trans (LGBT) people who - with the enthusiastic endorsement of bioethics - increasingly turn to assisted reproductive treatments to have children. In the face of these outcomes, it is better - according to this account - that people reconsider their interest in children. This account is not, however, ultimately strong enough to override people's interest in having children, relative to the benefits they confer and relative to the benefits conferred on children themselves. It is certainly not strong enough to justify differential treatment of LGBT people in matters of assisted reproductive treatments. Environmental threats in the wake of population growth might be managed in ways other than devaluing children as such. Moreover, this account ultimately damages the interests of LGBT people in matters of access, equity, and children, which outcome is paradoxical, given the origins of queer perspectives as efforts to assert and defend the social interests of sexual and gender minorities.

First Come, First Served in the Intensive Care Unit: Always?

Because the demand for intensive care unit (ICU) beds exceeds the supply in general, and because of the formidable costs of that level of care, clinicians face ethical issues when rationing this kind of care not only at the point of admission to the ICU, but also after the fact. Under what conditions-if any-may patients be denied admission to the ICU or removed after admission? One professional medical group has defended a rule of "first come, first served" in ICU admissions, and this approach has numerous moral considerations in its favor. We show, however, that admission to the ICU is not in and of itself guaranteed; we also show that as a matter of principle, it can be morally permissible to remove certain patients from the ICU, contrary to the idea that because they were admitted first, they are entitled to stay indefinitely through the point of recovery, death, or voluntary withdrawal. What remains necessary to help guide these kinds of decisions is the articulation of clear standards for discontinuing intensive care, and the articulation of these standards in a way consistent with not only fiduciary and legal duties that attach to clinical care but also with democratic decision making processes.

Immunoglobulin A Protease Variants Facilitate Intracellular Survival in Epithelial Cells By Nontypeable Haemophilus influenzae That Persist in the Human Respiratory Tract in Chronic Obstructive Pulmonary Disease.

Background: Nontypeable Haemophilus influenzae (NTHi) persists in the airways in chronic obstructive pulmonary disease (COPD). NTHi expresses 4 immunoglobulin (Ig)A protease variants (A1, A2, B1, B2) with distinct cleavage specificities for human IgA1. Little is known about the different roles of IgA protease variants in NTHi infection. Methods: Twenty-six NTHi isolates from a 20-year longitudinal study of COPD were analyzed for IgA protease expression, survival in human respiratory epithelial cells, and cleavage of lysosomal-associated membrane protein 1 (LAMP1). Results: IgA protease B1 and B2-expressing strains showed greater intracellular survival in host epithelial cells than strains expressing no IgA protease (P < .001) or IgA protease A1 or A2 (P < .001). Strains that lost IgA protease expression showed reduced survival in host cells compared with the same strain that expressed IgA protease B1 (P = .006) or B2 (P = .015). IgA proteases B1 and B2 cleave LAMP1. Passage of strains through host cells selected for expression of IgA proteases B1 and B2 but not A1. Conclusions: IgA proteases B1 and B2 cleave LAMP1 and mediate intracellular survival in respiratory epithelial cells. Intracellular persistence of NTHi selects for expression of IgA proteases B1 and B2. The variants of NTHi IgA proteases play distinct roles in pathogenesis of infection.

Antimicrobial activity of antisense peptide-peptide nucleic acid conjugates against non-typeable Haemophilus influenzae in planktonic and biofilm forms.

BACKGROUND: Antisense peptide nucleic acids (PNAs) are synthetic polymers that mimic DNA/RNA and inhibit bacterial gene expression in a sequence-specific manner. METHODS: To assess activity against non-typeable Haemophilus influenzae (NTHi), we designed six PNA-peptides that target acpP, encoding an acyl carrier protein. MICs and minimum biofilm eradication concentrations (MBECs) were determined. Resistant strains were selected by serial passages on media with a sub-MIC concentration of acpP-PNA. RESULTS: The MICs of six acpP-PNA-peptides were 2.9-11 mg/L (0.63-2.5 µmol/L) for 20 clinical isolates, indicating susceptibility of planktonic NTHi. By contrast, MBECs were up to 179 mg/L (40 µmol/L). Compared with one original PNA-peptide (acpP-PNA1-3'N), an optimized PNA-peptide (acpP-PNA14-5'L) differs in PNA sequence and has a 5' membrane-penetrating peptide with a linker between the PNA and peptide. The optimized PNA-peptide had an MBEC ranging from 11 to 23 mg/L (2.5-5 µmol/L), indicating susceptibility. A resistant strain that was selected by the original acpP-PNA1-3'N had an SNP that introduced a stop codon in NTHI0044, which is predicted to encode an ATP-binding protein of a conserved ABC transporter. Deletion of NTHI0044 caused resistance to the original acpP-PNA1-3'N, but showed no effect on susceptibility to the optimized acpP-PNA14-5'L. The WT strain remained susceptible to the optimized PNA-peptide after 30 serial passages on media containing the optimized PNA-peptide. CONCLUSIONS: A PNA-peptide that targets acpP, has a 5' membrane-penetrating peptide and has a linker shows excellent activity against planktonic and biofilm NTHi and is associated with a low risk for induction of resistance.