The lower airway microbiome in paediatric health and chronic disease.

The advent of next generation sequencing has rapidly challenged the paediatric respiratory physician's understanding of lung microbiology and the role of the lung microbiome in host health and disease. In particular, the role of "microbial key players" in paediatric respiratory disease is yet to be fully explained. Accurate profiling of the lung microbiome in children is challenging since the ability to obtain lower airway samples coupled with processing "low-biomass specimens" are both technically difficult. Many studies provide conflicting results. Early microbiota-host relationships may be predictive of the development of chronic respiratory disease but attempts to correlate lower airway microbiota in premature infants and risk of developing bronchopulmonary dysplasia (BPD) have produced mixed results. There are differences in lung microbiota in asthma and cystic fibrosis (CF). The increased abundance of oral taxa in the lungs may (or may not) promote disease processes in asthma and CF. In CF, correlation between microbiota diversity and respiratory decline is commonly observed. When one considers other pathogens beyond the bacterial kingdom, the contribution and interplay of fungi and viruses within the lung microbiome further increase complexity. Similarly, the interaction between microbial communities in different body sites, such as the gut-lung axis, and the influence of environmental factors, including diet, make the co-existence of host and microbes ever more complicated. Future, multi-omics approaches may help uncover novel microbiome-based biomarkers and therapeutic targets in respiratory disease and explain how we can live in harmony with our microbial companions.

Mepolizumab in children and adolescents with severe eosinophilic asthma not eligible for omalizumab: a single Center experience.

BACKGROUND: Mepolizumab is an anti-interleukin-5 monoclonal antibody shown to reduce asthma exacerbations in adults and adolescents with severe eosinophilic asthma. AIM: To assess the impact of mepolizumab on children and adolescents over 12 months by examining steroid usage, asthma-related hospitalizations, Asthma Control Test (ACT) scores, fractional exhaled nitric oxide concentration (FeNO), forced expiratory volume in 1 s (FEV1), mid expiratory flow (FEF25-75%), and blood eosinophil count. METHODS: Retrospective analysis performed between October 2015 and December 2022. Data was reviewed 12 months before and after commencing mepolizumab. Mepolizumab was offered if the patient had severe eosinophilic asthma and were unresponsive to or ineligible for omalizumab. RESULTS: Sixteen participants (age 7-17, 8 males, 8 females) received subcutaneous mepolizumab monthly with no serious adverse reactions. Incidence of hospital admissions fell significantly (IRR 0.33, p = 0.007). Among the 11 patients receiving daily oral corticosteroids, 3 were weaned off daily oral steroids and 3 patients' daily dose was significantly reduced (mean Δ-0.095 ± 0.071 mg/kg, p = 0.0012). Eosinophil count was decreased (mean Δ-0.85 x 109/L, p < 0.001). There was no significant change in mean overall steroid burden per patient (mean Δ-1445.63 ± 1603.18 mg, p = 0.10), ACT scores (mean Δ2.88 ± 6.71, p = 0.17), FEV1 z-scores (mean Δ-0.99 ± 1.88, p = 0.053), FEF25-75% z-scores (mean Δ-0.65 ± 1.61, p = 0.13), FeNO (mean Δ-20.09 ± 80.86, p = 0.34), or number of courses of oral steroids given for asthma attacks (IRR 0.71, p = 0.09). CONCLUSION: Among children and adolescents with severe eosinophilic asthma ineligible for or not responsive to omalizumab, mepolizumab therapy exhibited significant reduction in rate of asthma-related hospitalizations and significant decrease in daily steroid dosage.

The potential of bacteriophage therapy in the treatment of paediatric respiratory infections.

The looming antibiotic resistance crisis is forcing clinicians to consider alternative approaches to treating bacterial infections. As the window of use for current antimicrobial agents becomes ever narrower, we consider if looking back will now be the way forward. Conceptually, phage therapy is simple and specific; a targeted treatment to control bacterial overgrowth. In this article we discuss bacteriophage and potential use in future therapy.

Florida Red Tide: Inhalation Toxicity of Karenia brevis Extract in Rats.

Brevetoxins are neurotoxins produced by the marine dinoflagellate Karenia brevis. Histopathologic examination of marine mammals dying following repeated exposure of brevetoxins during red tide events suggests that the respiratory tract, nervous, hematopoietic, and immune systems are potential targets for toxicity in repeatedly exposed individuals. The purpose of this experiment was to evaluate the effects of repeated inhalation of K. brevis extract on these potential target systems in rats. Male Sprague-Dawley rats were exposed four hours/day, five days/week for up to four weeks to target concentrations of 200 and 1000 µg/L K. brevis extract (approximately 50 and 200 µg/L brevetoxin-like compounds; positive neurotoxicity in a fish bioassay). Control rats were sham exposed to air. Immunohistochemical staining of pulmonary macrophages indicated deposition of brevetoxin-like compound within the lung. However, exposure resulted in no clinical signs of toxicity or behavioral changes. There were no adverse effects on hematology or serum chemistry. No histopathological changes were observed in the nose, lung, liver, kidneys, lymph nodes, spleen, or brain of exposed rats. Immune suppression was suggested by reduced responses of spleen cells in the IgM-specific antibody-forming plaque cell response assay and reduced responses of lymphocytes to mitogen stimulation in vitro. Differences between responses observed in rats in this study and those observed in manatees may be a function of dose or species differences in sensitivity.

Response of F344 rats to inhalation of subclinical levels of sarin: exploring potential causes of Gulf War illness.

Subclinical, repeated exposures of F344 rats to sarin resulted in brain alterations in densities of chlonergic receptor subtypes that may be associated with memory loss and cognitive dysfunction. The exposures also depressed the immune system. The rat appears to be a good model for studying the effects of subclinical exposure to a nerve gas.

HIV type-1 infection of the cotton rat (Sigmodon fulviventer and S. hispidus).

Cotton rats (Sigmodon hispidus and S. fulviventer) are susceptible to many viruses that infect humans (e.g., poliovirus, respiratory syncytial virus, influenza virus, adenovirus, and parainfluenza virus) and have been influential in developing therapeutic clinical intervention strategies for many viral infections of man. This study set out to determine whether cotton rats are susceptible to infection with HIV type 1 (HIV-1). Results indicate that HIV-1 does infect the cotton rat and S. fulviventer is more susceptible than S. hispidus. The virus was passaged from animal to animal for a total of three serial passages; but HIV replicated poorly in vivo, was only detectable as proviral DNA, and never exceeded one provirus per 1.8 x 10(5) cotton rat peripheral blood mononuclear cells. Infection induced a distinct and characteristic anti-HIV antibody response that, in some animals, included neutralizing antibodies, recognized all of the major HIV-1 antigens and the antibodies lasted out to 52 wk post-infection. Neonate S. fulviventer were not more susceptible to infection than adults. In vitro culture studies produced indirect evidence of viral replication by detection of viral gag gene RNA in reverse transcriptase-PCR assays on viral culture supernatants. Collectively, these results indicate that HIV-1 can replicate in a nontransgenic rodent and that this system may have potential as an animal model for HIV-1 infection if viral replication rates can be improved in vivo.

Nucleotide sequence analysis of puma lentivirus (PLV-14): genomic organization and relationship to other lentiviruses.

The complete nucleotide sequence of an isolate of puma lentivirus (PLV-14) was obtained by an inverse polymerase chain reaction (I-PCR) technique and confirmed by conventional PCR. Both methods were used to amplify overlapping regions of proviral DNA, for cloning and sequencing, from genomic DNA isolated from PLV-14 infected Florida puma (Felis concolor coryi) peripheral blood mononuclear cells (PBMC). The provirus has a total length of 9100 nucleotides and the genomic organization of presumed protein coding regions are similar to those seen in other members of the lentivirus family, i.e., three large open reading frames gag, pol, and env as well as smaller intergenic regions that apparently encode regulatory proteins vif and 3' rev by positional and sequence similarity to those seen in other lentiviruses. Two additional open reading frames were identified in the env region and their function (if any) is unknown. The length of the PLV-14 long terminal repeat (LTR) was found to be shorter than the LTRs of feline immunodeficiency virus (FIV). The sequence homology between PLV-14 and other lentiviruses demonstrates that PLV-14 is most closely related to FIV from domestic cats. However, the extent of sequence divergence of each retroviral gene segment is large (e.g., percentage sequence similarity between FIV and PLV-14 env is 8% amino acid and 37% nucleotide similarity), indicating relatively ancient divergence of these feline lentiviral genomes.

An idiotypic marker associated with a germ-line encoded kappa light chain variable region that predominates the vaccine-induced human antibody response to the Haemophilus influenzae b polysaccharide.

Human antibodies specific for the Haemophilus influenzae b polysaccharide (Hib PS) frequently express a cross-reactive idiotype (CRI), and commonly utilize a VL region that is the product of the V kappa II gene A2. To examine further anti-Hib PS V region expression and to determine whether CRI expression is correlated with the V kappa IIA2 chain, we isolated a monoclonal antibody (MAb) reactive with an idiotypic determinant of anti-Hib PS antibodies. This MAb inhibited Hib PS binding but did not react with Ig isotypic determinants. The CRI recognized by this MAb, designated HibId-1, was associated with the Hib PS-combining site since the reactivity of the MAb with anti-Hib PS antibodies could be inhibited by Hib PS. HibId-1 was expressed by 17 of 17 clonally purified and sequence-defined anti-Hib PS antibodies having V kappa IIA2 L chains. In contrast, 0 of 10 anti-Hib PS antibodies having either V lambda, V kappa I, or V kappa III chains expressed HibId-1. Western blot analysis showed that the MAb anti-CRI reacted with isolated anti-Hib PS V kappa IIA2 L chains but not with H chains or other L chains, indicating that the HibId-1 determinant is localized to the V kappa IIA2 chain, and does not require pairing with H chain for expression. Anti-Hib PS antibodies bearing HibId-1 were present in at least 85% of subjects immunized with either free Hib PS or Hib PS coupled to diphtheria toxoid (Hib PS-DT), and comprised on the average 60% of the total vaccine-induced serum anti-Hib PS. HibId-1 expression was not related to age at vaccination inasmuch as infants, children, and adults had similar distributions of HibId-1-positive anti-Hib PS after vaccination with Hib PS-DT. HibId-1 was expressed at a lower frequency and comprised a smaller fraction of the total anti-Hib PS antibody in adult preimmunization sera as compared to post-Hib PS immunization sera, suggesting that immunization preferentially stimulates HibId-1-positive B cells. These data demonstrate that antibodies bearing HibId-1/V kappa IIA2 comprise a predominant component of the anti-Hib PS response induced by immunization, and that this pattern of VL expression is established early in ontogeny.

Detection of circulating immune complexes by the enzyme-linked immunosorbent assay in sera from cattle infected with Fasciola hepatica.

Polyethylene glycol-precipitated immune complexes (PIC) from the sera of 5 calves with Fasciola hepatica worm burdens ranging between 27 and 70 flukes were examined for parasite antigen content at 2, 4, 6, 8, 10, and 16 wk postinfection (PI) by the enzyme-linked immunosorbent assay (ELISA). Three assays were devised using an affinity-processed rabbit antibody to worm excretory/secretory (FhES) antigens. The PIC plate assay detected parasite antigen by adherence of anti-FhES antibody to PIC incubated overnight on ELISA plates, and tests were visualized using anti-rabbit peroxidase-linked antibody. The serum complex and PIC capture assay utilized the anti-FhES immunoglobulin as an antigen capture antibody linked to the solid phase. The attached complexes were then detected by the adhering bovine antibody, either soluble complexes in serum or as PIC. All assays showed circulating immune complex (CIC) values elevated at 6-8 wk PI, which generally coincided with increased host circulating antibody to FhES antigens. The greatest detection rate for all of the immune complex (IC) detection assays occurred with the PIC capture assay. It detected antigen in almost 90% of sera tested at 6 and 8 wk PI. Both the serum complex and PIC capture assay detected greatest amounts of CIC in those animals with the largest worm burdens, whereas the PIC plate assay showed no such trend. This study shows that F. hepatica antigen detection in CIC can be used to aid immunologic diagnosis of fascioliasis.

Detection of circulating parasite antigen in murine fascioliasis by two-site enzyme-linked immunosorbent assays.

A 2-site enzyme immunoassay was developed for the detection of Fasciola hepatica antigen in the serum of fascioliasis infected mice. The assay utilizes high titer rabbit immunoglobulins to parasite excretory/secretory antigens (FhES) as capture antibody, and also as detection antibody when linked to horseradish peroxidase (HRP) or to biotin for reaction with avidin-peroxidase. The assays were compared with a conventional (antibody detection) ELISA to determine diagnostic utility. Using mean rates of detection of fascioliasis, the HRP-based antigen capture assay diagnosed the infection at 1 week postinfection and showed that circulating antigen levels are maximal 3 weeks after infection. The earliest mean diagnosis for the antibody detection and the biotin-based antigen capture ELISAs were 2 and 3 weeks postinfection, respectively. The addition of known quantities of FhES antigens to normal mouse serum gave estimates of lower limits of detectability for the HRP- and biotin-based assays of 25 ng and 0.25 ng, respectively. Routine use of the biotin-avidin system in the antigen capture test resulted in high background activity making this method insensitive.

Vaccination against bovine babesiosis with drug-controlled live parasites.

Live Babesia divergens derived from gerbils were used to vaccinate cattle that had previously been treated with imidocarb dipropionate. Drug doses ranged from 1 to 2 mg/kg and animals were infected subcutaneously three to seven days later. After a further 35 days, vaccinated and control animals were given a heavy heterologous intravenous challenge. This regimen was effective for both avirulent and virulent strains in 12- to 18-month-old cattle. However, at low drug doses some animals reacted to the virulent vaccine strain and at high doses animals infected with the avirulent strain failed to seroconvert although they were still resistant to challenge. The variable infectivity of vaccine strains was a minor problem which can be overcome by strain selection and optimisation of infectivity using gerbils as experimental animals. Gerbils would also be useful as a source of parasites for the further development of in vitro cultures which could ultimately produce the vaccine.

Non-specific resistance to Babesia divergens in the Mongolian gerbil (Meriones unguiculatus).

Inoculation of mature gerbils with BCG gave protection to subsequent infection with B. divergens when inoculated by the intracardiac and intraperitoneal routes, the latter showing a dose dependent relationship. BCG vaccination was most effective in immature gerbils (less than 4 weeks old), which are innately resistant to B. divergens. Vaccination of gerbils with killed Propionesbacterium acne and zymosan A failed to elicit a protective response, which contrasts conspicuously with rodent babesia studies. Incubation of B. divergens-infected gerbil blood with hydrogen peroxide produced parasite inhibition only at the highest concentration and treatment of parasitized gerbils with the oxidative radical inducer, alloxan monohydrate, gave equivocal results so it is evident that, unlike Plasmodium spp., B. divergens is not significantly susceptible to the action of reactive oxygen forms.

Age-related susceptibility of the gerbil, Meriones unguiculatus, to the bovine parasite, Babesia divergens.

Immature gerbils (4 weeks of age or less) proved to be more resistant to Babesia divergens infections than did mature animals (greater than 8 weeks old). Nutritional studies showed that a milk diet contributes little to this innate resistance. Similarly, the apparent predilection of B. divergens for mature erythrocytes, and also in vitro serum incubation studies, suggested that blood factors do not contribute to any major degree. Splenectomy of immature gerbils abolished their nonspecific resistance and studies with splenocyte incubations with the piroplasm imply that splenic integrity is necessary for the maintenance of this, apparently antibody-independent, resistance to infection with B. divergens.

Effects of rapid passage in the gerbil (Meriones unguiculatus) on the course of infection of the bovine piroplasm Babesia divergens in splenectomised calves.

The antigenicity and virulence of the cattle piroplasm Babesia divergens was compared in splenectomised calves before and after adaptation to the gerbil. It was apparent that adaptation of B divergens to the gerbil did not result in attenuation of the parasite in cattle. The gerbil-adapted parasite had a shorter prepatent period, produced higher parasitaemias, and more severe haematological and biochemical changes than the initial bovine strain. The antigenicity of the parasite was unchanged by gerbil passage. The changes in the virulence of the gerbil-adapted parasite were attributed to selection of a rapidly replicating parasite.

Morphological comparisons of the bovine piroplasm, Babesia divergens, in cattle and jird (Meriones unguiculatus) erythrocytes.

In comparative studies of Babesia divergens in jirds and splenectomized calves several differences in the appearance of the parasite were found. Pyriform pairs of the parasite grew larger in jirds than in calves and also were larger than those of the original isolate when calves were infected after multiple jird passage. The parasites usually occupied a central intraerythrocytic position in jirds whereas in cattle they mainly occurred peripherally. The majority of pairs formed an obtuse angle relative to each other in both host species. The main difference in morphological types was found to be the rarity of single pyriforms in jirds and this, together with other small differences, was attributed to a faster replication rate of B. divergens in this host. Intraerythrocytic death of B. divergens was observed in both jirds and calves indicating similar defence mechanisms against primary infections in the 2 host species.