Respiratory mucosal delivery of next-generation COVID-19 vaccine provides robust protection against both ancestral and variant strains of SARS-CoV-2.

The emerging SARS-CoV-2 variants of concern (VOCs) threaten the effectiveness of current COVID-19 vaccines administered intramuscularly and designed to only target the spike protein. There is a pressing need to develop next-generation vaccine strategies for broader and long-lasting protection. Using adenoviral vectors (Ad) of human and chimpanzee origin, we evaluated Ad-vectored trivalent COVID-19 vaccines expressing spike-1, nucleocapsid, and RdRp antigens in murine models. We show that single-dose intranasal immunization, particularly with chimpanzee Ad-vectored vaccine, is superior to intramuscular immunization in induction of the tripartite protective immunity consisting of local and systemic antibody responses, mucosal tissue-resident memory T cells and mucosal trained innate immunity. We further show that intranasal immunization provides protection against both the ancestral SARS-CoV-2 and two VOC, B.1.1.7 and B.1.351. Our findings indicate that respiratory mucosal delivery of Ad-vectored multivalent vaccine represents an effective next-generation COVID-19 vaccine strategy to induce all-around mucosal immunity against current and future VOC.

The transient receptor potential vanilloid 4 (TRPV4) ion channel mediates protease activated receptor 1 (PAR1)-induced vascular hyperpermeability.

Endothelial barrier disruption is a hallmark of tissue injury, edema, and inflammation. Vascular endothelial cells express the G protein-coupled receptor (GPCR) protease acctivated receptor 1 (PAR1) and the ion channel transient receptor potential vanilloid 4 (TRPV4), and these signaling proteins are known to respond to inflammatory conditions and promote edema through remodeling of cell-cell junctions and modulation of endothelial barriers. It has previously been established that signaling initiated by the related protease activated receptor 2 (PAR2) is enhanced by TRPV4 in sensory neurons and that this functional interaction plays a critical role in the development of neurogenic inflammation and nociception. Here, we investigated the PAR1-TRPV4 axis, to determine if TRPV4 plays a similar role in the control of edema mediated by thrombin-induced signaling. Using Evans Blue permeation and retention as an indication of increased vascular permeability in vivo, we showed that TRPV4 contributes to PAR1-induced vascular hyperpermeability in the airways and upper gastrointestinal tract of mice. TRPV4 contributes to sustained PAR1-induced Ca2+ signaling in recombinant cell systems and to PAR1-dependent endothelial junction remodeling in vitro. This study supports the role of GPCR-TRP channel functional interactions in inflammatory-associated changes to vascular function and indicates that TRPV4 is a signaling effector for multiple PAR family members.

The addition of Lactobacillus spp. negatively affects Mycoplasma bovis viability in bovine cervical mucus.

BACKGROUND: Mycoplasma bovis is an important pathogen for the cattle industry worldwide causing significant economic losses. Several transmission routes, including those related to reproduction, have been described. Indeed, the pathogen can colonize the female reproductive tract after artificial insemination (AI) with contaminated semen. Lactobacillus spp.-based probiotics have been used for vaginal dysbiosis treatment in women and cows although their role in controlling cervico-vaginal infections due to M. bovis is unknown. The objective of the present work is to assess the viability of M. bovis (PG45, NCTC 10131) in experimentally contaminated cervical mucus after the addition of Lactobacillus spp. at different concentrations as a competing agent and pH acidifier. RESULTS: The addition of probiotic at a concentration higher than 108 colony forming units (CFU/mL had a detrimental effect (P < 0.05) on mycoplasma viability in cervical mucus. This coincided with a significant LAB growth and an important decrease in pH from 8.4 to 5.6 (P < 0.05). However, after the addition of less concentrated probiotic, M. bovis survival was not affected and there was no significant LAB growth despite the drop of pH from 8.4 to 6.73 (P < 0.05). CONCLUSION: The addition of concentrations higher than 108 CFU/mL of Lactobacillus spp. negatively affects M. bovis viability in bovine cervical mucus under in vitro conditions. Although the effect observed on the pathogen viability seems to be related to the pH decrease after LAB proliferation in cervical mucus, further studies are necessary to elucidate if other factors are implicated. Nevertheless, the administration of Lactobacillus spp.-based probiotics might be used in the future to control M. bovis proliferation in the cervico-vaginal tract of cows.

Assessment of in vitro growth characteristics of Mycoplasma ovipneumoniae.

Mycoplasma ovipneumoniae (Movp) is an emerging pathogen that causes respiratory disease in small ruminants worldwide. It is considered to be difficult and time consuming to grow, which complicates diagnostic and control measures including isolation (an essential step required prior to the characterisation of strains), antimicrobial susceptibility testing and the development of vaccines. The objectives of this study were to analyse in vitro growth patterns of Movp strains, and the effects of different media used to support their growth. The study was conducted on 20 ovine and caprine Movp strains, isolated using Thiaucourt's medium. The rapid growth phase varied among the strains from 24 h to 72 h, although 60% of strains (12 of 20) reached a peak at 48 h. All strains were viable at 72 h after incubation, and declining viability was observed at 96 h (13 of 20 remained viable; 65%), 120 h (9 of 20; 45%) and 144 h (4 of 20; 20%). Growth was not detected at 168 h. All strains were able to grow in modified tryptone soy broth, while PH mycoplasma medium-Hayflick modified medium supported the growth of only two strains. Improved techniques of Movp cultivation require consideration of the growth variability among strains, the time of subculturing (during the first three days of incubation) and selection of appropriate media.

Mycoplasma ovipneumoniae (Movp) est un agent pathogène émergent qui provoque une maladie respiratoire chez les petits ruminants du monde entier. Sa croissance est lente et jugée difficile à obtenir, ce qui complique le diagnostic et les mesures de lutte, en particulier l'isolement (étape indispensable pour caractériser les souches), le recours aux antibiogrammes et le développement de vaccins. L'étude présentée par les auteurs a pour objectifs d'analyser in vitro les profils de croissance de différentes souches de Movp et d'observer l'effet sur cette croissance des milieux utilisés. L'étude a porté sur 20 souches de Movp provenant d'ovins et de caprins isolées sur milieu de Thiaucourt. La phase de croissance rapide variait de 24 h à 72 h suivant les souches, avec néanmoins un pic de croissance atteint en 48 h pour 12 des 20 souches (soit 60 %). La totalité des souches étaient viables 72 h après l'incubation ; une perte de la viabilité a été observée à 96 h (13 souches sur 20 demeurant viables, soit 65 %), à 120 h (9 souches viables sur 20, soit 45 %) et à 144 h (4 souches viables sur 20, soit 20 %). Aucun signe de croissance n'était détecté à 168 h. L'ensemble des souches ont présenté des signes de croissance sur bouillon tryptone soja modifié, tandis que deux souches seulement ont présenté des signes de croissance sur milieu PH mycoplasma-milieu de Hayflick modifié. L'amélioration des techniques de mise en culture des Movp passe par la prise en compte de la variabilité de la croissance suivant les souches, par un repiquage réalisé au bon moment (dans les trois premiers jours d'incubation) et par le choix approprié du milieu.

Mycoplasma ovipneumoniae (Movp) es un patógeno emergente que afecta a pequeños rumiantes del mundo entero, en los que provoca una enfermedad respiratoria. Su cultivo está considerado difícil y exige tiempo, lo que complica su diagnóstico y las medidas de lucha, en particular el aislamiento (paso previo indispensable para la caracterización de las cepas), la realización de pruebas de sensibilidad a los antimicrobianos y la obtención de vacunas. Los autores describen un estudio encaminado a analizar las modalidades de crecimiento in vitro de cepas de Movp y los efectos del uso de diferentes medios de cultivo, utilizando para ello 20 cepas ovinas y caprinas de Movp aisladas con empleo de medio de Thiaucourt. La fase de crecimiento rápido, variable en función de la cepa, iba de 24 a 72 horas, aunque un 60% de las cepas (12 de 20) alcanzaba el pico de crecimiento a las 48 horas. Todas las cepas eran viables a las 72 horas de incubación. Se observó asimismo que la viabilidad menguaba al cabo de 96 horas (seguían siendo viables 13 de las 20 cepas, un 65%), 120 horas (9 de 20, un 45%) y 144 horas (4 de 20, un 20%). Al cabo de 168 horas no se detectaba crecimiento alguno. Todas las cepas podían crecer en caldo de triptona de soja modificado, mientras que solo dos cepas crecían en un medio PH mycoplasma-medio Hayflick modificado. Para poder perfeccionar las técnicas de cultivo de Movp es preciso tener en cuenta la variabilidad entre las cepas por lo que respecta a las características de crecimiento, así como el tiempo de subcultivo (durante los tres primeros días de incubación) y la selección del medio apropiado.

Epidemiological role of birds in the transmission and maintenance of zoonoses. / Papel epidemiológico de las aves en la transmisión y mantenimiento de zoonosis.

The risk of zoonoses spreading from birds to humans is lower, quantitatively speaking, than the risk of transmission between other host groups, because the two taxonomic groups share fewer pathogens. Nevertheless, birds have a number of epidemiological characteristics that make them extremely important hosts in the transmission and maintenance of zoonoses, including their susceptibility to pathogens that are extremely hazardous to humans (such as highly pathogenic avian influenza virus, West Nile virus and Chlamydia psittaci) and their ability to travel long distances, especially in the case of migratory birds. The fact that the human diet includes poultry products (meat, eggs and their by-products) also means that most human cases of foodborne zoonoses are infections of avian origin. Lastly, close contact between humans and pet birds or urban birds leads to interactions of public health concern. This article sets out to describe the main factors that determine the role of birds in the epidemiology of zoonotic infections.

Le risque que les oiseaux transmettent des zoonoses à l'homme est moins élevé, au plan quantitatif, qu'entre hôtes d'autres catégories, car le nombre d'agents pathogènes affectant à la fois ces deux groupes taxonomiques est moindre. Cependant, certaines particularités épidémiologiques des oiseaux leur font jouer un rôle d'hôtes importants dans la persistance et la transmission de zoonoses : d'une part, leur sensibilité à des agents pathogènes dangereux pour l'homme (par exemple, le virus de l'influenza aviaire hautement pathogène, le virus de West Nile, Chlamydia psittaci) et, d'autre part, leur capacité à se déplacer sur de longues distances, notamment dans le cas des oiseaux migrateurs. En outre, les produits avicoles faisant partie des denrées alimentaires consommées par l'homme (viande de volaille, oeufs et produits dérivés), la majorité des cas de zoonoses d'origine alimentaire diagnostiqués chez l'homme sont d'origine aviaire. Enfin, les contacts étroits entre les humains et leurs oiseaux de compagnie ou avec des oiseaux des villes entraînent des interactions qui sont à prendre en compte en santé publique. Les auteurs décrivent les principales caractéristiques épidémiologiques des oiseaux jugées déterminantes par rapport aux infections zoonotiques.

El riesgo de transmisión de zoonosis de aves a humanos es menor, cuantitativamente hablando, que el que tiene lugar entre otros grupos de hospedadores, debido a que estos dos grupos taxonómicos comparten un menor número de agentes patógenos. No obstante, algunas particularidades epidemiológicas de las aves las convierten en hospedadores de gran importancia en el mantenimiento y la transmisión de zoonosis, como su capacidad de contraer infecciones por agentes patógenos peligrosos para los humanos (como el virus de la influenza aviar altamente patógena, el virus del Nilo Occidental o Chlamydia psittaci, entre otros) así como su gran capacidad de desplazamiento, especialmente en el caso de las aves migratorias. Además, el hecho de que la alimentación humana incluya productos avícolas (carne y huevos y productos derivados) hace que la mayoría de casos de zoonosis de origen alimentario diagnosticados en humanos sean infecciones de origen aviar. Por último, el estrecho contacto entre humanos y mascotas aviares o aves urbanas conlleva interacciones de interés para la salud pública. Este trabajo pretende describir los principales determinantes epidemiológicos de las aves en relación con las infecciones zoonósicas.

Sensitivity of two methods to detect Mycoplasma agalactiae in goat milk.

BACKGROUND: Laboratory diagnostic techniques able to detect Mycoplasma agalactiae are essential in contagious agalactia in dairy goats. This study was designed: 1) to determine the detection limits of PCR and culture in goat milk samples, 2) to examine the effects of experimental conditions including the DNA extraction method, PCR technique and storage conditions (fresh versus frozen stored milk samples) on these methods and 3), to establish agreement between PCR and culture techniques using milk samples from goats with mastitis in commercial dairy herds. The study was conducted both on artificially inoculated and field samples. RESULTS: Our findings indicate that culture is able to detect M. agalactiae in goat milk at lower concentrations than PCR. Qualitative detection of M.agalactiae by culture and PCR was not affected by sample freezing, though the DNA extraction method used significantly affected the results of the different PCR protocols. When clinical samples were used, both techniques showed good agreement. CONCLUSIONS: The results from this study indicate that both culture and PCR are able to detect M. agalactiae in clinical goat mastitis samples. However, in bulk tank milk samples with presumably lower M. agalactiae concentrations, culture is recommended within the first 24 h of sample collection due to its lower limit of detection. To improve the diagnostic sensitivity of PCR in milk samples, there is a need to increase the efficiency of extracting DNA from milk samples using protocols including a previous step of enzymatic digestion.

Protease-activated receptor 2 (PAR2) protein and transient receptor potential vanilloid 4 (TRPV4) protein coupling is required for sustained inflammatory signaling.

G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR(2)), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR(2) regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR(2) agonist stimulated a transient increase in [Ca(2+)](i). TRPV4 expression led to a markedly sustained increase in [Ca(2+)](i). Removal of extracellular Ca(2+) and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A(2) and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR(2) generates arachidonic acid-derived lipid mediators, such as 5',6'-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR(2)-induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR(2) was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR(2) signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR(2)-stimulated neurogenic inflammation. Thus, PAR(2) activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR(2).

Facilitation of macrofaunal assemblages in marinas by the habitat-forming invader Amathia verticillata (Bryozoa: Gymnolaemata) across a spatiotemporal scale.

Widespread habitat-forming invaders inhabiting marinas, such as the spaghetti bryozoan Amathia verticillata, allow exploring facilitation processes across spatiotemporal contexts. Here we investigate the role of this bryozoan as habitat for native and exotic macrofaunal assemblages across different ecoregions of Western Mediterranean and East Atlantic coasts, and a monthly variation over a year. While only 7 (all peracarid crustaceans) of the 54 associated species were NIS, they dominated macrofaunal assemblages in terms of abundance, raising the potential for invasional meltdown. NIS richness and community structure differed among marinas but not among ecoregions, highlighting the importance of marina singularities in modulating facilitation at spatial scale. Despite facilitation did not depend on bryozoan abundance fluctuations, it was affected by its deciduous pattern, peaking in summer and disappearing in late winter. Monitoring A. verticillata in marinas, especially in summer periods, may improve the detection and management of multiple associated NIS.

Comparison of "IN-REC-SUR-E" and LISA in preterm neonates with respiratory distress syndrome: a randomized controlled trial (IN-REC-LISA trial).

BACKGROUND: Surfactant is a well-established therapy for preterm neonates affected by respiratory distress syndrome (RDS). The goals of different methods of surfactant administration are to reduce the duration of mechanical ventilation and the severity of bronchopulmonary dysplasia (BPD); however, the optimal administration method remains unknown. This study compares the effectiveness of the INtubate-RECruit-SURfactant-Extubate (IN-REC-SUR-E) technique with the less-invasive surfactant administration (LISA) technique, in increasing BPD-free survival of preterm infants. This is an international unblinded multicenter randomized controlled study in which preterm infants will be randomized into two groups to receive IN-REC-SUR-E or LISA surfactant administration. METHODS: In this study, 382 infants born at 24+0-27+6 weeks' gestation, not intubated in the delivery room and failing nasal continuous positive airway pressure (nCPAP) or nasal intermittent positive pressure ventilation (NIPPV) during the first 24 h of life, will be randomized 1:1 to receive IN-REC-SUR-E or LISA surfactant administration. The primary outcome is a composite outcome of death or BPD at 36 weeks' postmenstrual age. The secondary outcomes are BPD at 36 weeks' postmenstrual age; death; pulse oximetry/fraction of inspired oxygen; severe intraventricular hemorrhage; pneumothorax; duration of respiratory support and oxygen therapy; pulmonary hemorrhage; patent ductus arteriosus undergoing treatment; percentage of infants receiving more doses of surfactant; periventricular leukomalacia, severe retinopathy of prematurity, necrotizing enterocolitis, sepsis; total in-hospital stay; systemic postnatal steroids; neurodevelopmental outcomes; and respiratory function testing at 24 months of age. Randomization will be centrally provided using both stratification and permuted blocks with random block sizes and block order. Stratification factors will include center and gestational age (24+0 to 25+6 weeks or 26+0 to 27+6 weeks). Analyses will be conducted in both intention-to-treat and per-protocol populations, utilizing a log-binomial regression model that corrects for stratification factors to estimate the adjusted relative risk (RR). DISCUSSION: This trial is designed to provide robust data on the best method of surfactant administration in spontaneously breathing preterm infants born at 24+0-27+6 weeks' gestation affected by RDS and failing nCPAP or NIPPV during the first 24 h of life, comparing IN-REC-SUR-E to LISA technique, in increasing BPD-free survival at 36 weeks' postmenstrual age of life. TRIAL REGISTRATION: ClinicalTrials.gov NCT05711966. Registered on February 3, 2023.

N-glycosylation determines ionic permeability and desensitization of the TRPV1 capsaicin receptor.

The balance of glycosylation and deglycosylation of ion channels can markedly influence their function and regulation. However, the functional importance of glycosylation of the TRPV1 receptor, a key sensor of pain-sensing nerves, is not well understood, and whether TRPV1 is glycosylated in neurons is unclear. We report that TRPV1 is N-glycosylated and that N-glycosylation is a major determinant of capsaicin-evoked desensitization and ionic permeability. Both N-glycosylated and unglycosylated TRPV1 was detected in extracts of peripheral sensory nerves by Western blotting. TRPV1 expressed in HEK-293 cells exhibited various degrees of glycosylation. A mutant of asparagine 604 (N604T) was not glycosylated but did not alter plasma membrane expression of TRPV1. Capsaicin-evoked increases in intracellular calcium ([Ca(2+)](i)) were sustained in wild-type TRPV1 HEK-293 cells but were rapidly desensitized in N604T TRPV1 cells. There was marked cell-to-cell variability in capsaicin responses and desensitization between individual cells expressing wild-type TRPV1 but highly uniform responses in cells expressing N604T TRPV1, consistent with variable levels of glycosylation of the wild-type channel. These differences were also apparent when wild-type or N604T TRPV1-GFP fusion proteins were expressed in neurons from trpv1(-/-) mice. Capsaicin evoked a marked, concentration-dependent increase in uptake of the large cationic dye YO-PRO-1 in cells expressing wild-type TRPV1, indicative of loss of ion selectivity, that was completely absent in cells expressing N604T TRPV1. Thus, TRPV1 is variably N-glycosylated and glycosylation is a key determinant of capsaicin regulation of TRPV1 desensitization and permeability. Our findings suggest that physiological or pathological alterations in TRPV1 glycosylation would affect TRPV1 function and pain transmission.

Invasive sino-orbital mycosis in an aplastic anemia patient caused by Neosartorya laciniosa.

We report the first case of Neosartorya laciniosa invasive sinusitis involving the orbit in an immunocompromised male with aplastic anemia. Treatment included surgical debridement with enucleation of the eye and combination voriconazole and micafungin therapy followed by voriconazole alone. The fungus was identified using sequencing of partial benA and calmodulin genes.

Short communication: In vitro antimicrobial susceptibility of Mycoplasma agalactiae strains isolated from dairy goats.

This study examined the susceptibility to several antimicrobials of 28 isolates of Mycoplasma agalactiae obtained from goats in a region (southeastern Spain) where contagious agalactia is endemic. For each isolate, the minimum inhibitory concentration (MIC) against 12 antimicrobials of the quinolone, macrolide, aminoglycoside, and tetracycline families was determined. The antimicrobials with the lowest MIC were enrofloxacin, ciprofloxacin, tylosin, and doxycycline, all with MIC90 (concentration at which growth of 90% of the isolates is inhibited) <1 µg/mL. Norfloxacin (a quinolone) showed a wide MIC range (0.1-12.8 µg/mL), suggesting a resistance mechanism toward this antimicrobial that was not elicited by enrofloxacin or ciprofloxacin (the other quinolones tested). Erythromycin showed the highest MIC90 such that its use against Mycoplasma agalactiae is not recommended. Finally, Mycoplasma agalactiae isolates obtained from goat herds with clinical symptoms of contagious agalactia featured higher MIC90 and MIC50 (concentration at which growth of 50% of the isolates is inhibited) values for many of the antimicrobials compared with isolates from asymptomatic animals. The relationship between the extensive use of antimicrobials in herds with clinical contagious agalactia and variations in MIC requires further study.

Ligand affinity and kinase activity are independent of bacterial chemotaxis receptor concentration: insight into signaling mechanisms.

Binding of attractant to bacterial chemotaxis receptors initiates a transmembrane signal that inhibits the kinase CheA bound ~300 Å distant at the other end of the receptor. Chemoreceptors form large clusters in many bacterial species, and the extent of clustering has been reported to vary with signaling state. To test whether ligand binding regulates kinase activity by modulating a clustering equilibrium, we measured the effects of two-dimensional receptor concentration on kinase activity in proteoliposomes containing the purified Escherichia coli serine receptor reconstituted into vesicles over a range of lipid:protein molar ratios. The IC(50) of kinase inhibition was unchanged despite a 10-fold change in receptor concentration. Such a change in concentration would have produced a measurable shift in the IC(50) if receptor clustering were involved in kinase regulation, based on a simple model in which the receptor oligomerization and ligand binding equilibria are coupled. These results indicate that the primary signal, ligand control of kinase activity, does not involve a change in receptor oligomerization state. In combination with previous work on cytoplasmic fragments assembled on vesicle surfaces [Besschetnova, T. Y., et al. (2008) Proc. Natl. Acad. Sci. U.S.A.105, 12289-12294], this suggests that binding of ligand to chemotaxis receptors inhibits the kinase by inducing a conformational change that expands the membrane area occupied by the receptor cytoplasmic domain, without changing the number of associated receptors in the signaling complex.

Sites of action of ghrelin receptor ligands in cardiovascular control.

Circulating ghrelin reduces blood pressure, but the mechanism for this action is unknown. This study investigated whether ghrelin has direct vasodilator effects mediated through the growth hormone secretagogue receptor 1a (GHSR1a) and whether ghrelin reduces sympathetic nerve activity. Mice expressing enhanced green fluorescent protein under control of the promoter for growth hormone secretagogue receptor (GHSR) and RT-PCR were used to locate sites of receptor expression. Effects of ghrelin and the nonpeptide GHSR1a agonist capromorelin on rat arteries and on transmission in sympathetic ganglia were measured in vitro. In addition, rat blood pressure and sympathetic nerve activity responses to ghrelin were determined in vivo. In reporter mice, expression of GHSR was revealed at sites where it has been previously demonstrated (hypothalamic neurons, renal tubules, sympathetic preganglionic neurons) but not in any artery studied, including mesenteric, cerebral, and coronary arteries. In rat, RT-PCR detected GHSR1a mRNA expression in spinal cord and kidney but not in the aorta or in mesenteric arteries. Moreover, the aorta and mesenteric arteries from rats were not dilated by ghrelin or capromorelin at concentrations >100 times their EC(50) determined in cells transfected with human or rat GHSR1a. These agonists did not affect transmission from preganglionic sympathetic neurons that express GHSR1a. Intravenous application of ghrelin lowered blood pressure and decreased splanchnic nerve activity. It is concluded that the blood pressure reduction to ghrelin occurs concomitantly with a decrease in sympathetic nerve activity and is not caused by direct actions on blood vessels or by inhibition of transmission in sympathetic ganglia.

Evaluation of Host Cell Impurity Effects on the Performance of Sterile Filtration Processes for Therapeutic Viruses.

Efficient downstream processing represents a significant challenge in the rapidly developing field of therapeutic viruses. While it is known that the terminal sterile filtration step can be a major cause of product loss, there is little known about the effect of host cell impurities (DNA and protein) on filtration performance. In this study, fractions of relatively pure Vero host cell protein and DNA were spiked into a highly pure preparation of vesicular stomatitis virus (VSV). Then, the resulting solutions were sterile filtered using two commercially available 0.22 µm rated microfiltration membranes. A combination of transmembrane pressure measurements, virus recovery measurements, and post-filtration microscopy images of the microfiltration membranes was used to evaluate the sterile filtration performance. It was found that increasing the amount of host cell protein from approximately 1 µg/mL (in the un-spiked VSV preparation) to 25 µg/mL resulted in a greater extent of membrane fouling, causing the VSV recovery to decrease from 89% to 65% in experiments conducted with the highly asymmetric Express PLUS PES membrane and to go as low as 48% in experiments conducted with the symmetric Durapore PVDF membrane. Similar effects were not seen when bovine serum albumin, a common model protein used in filtration studies, was spiked into the VSV preparation, which indicates that the sterile filtration performance is critically dependent on the complex composition of the mixture of host cell proteins rather than the presence of any protein. The results presented in this work provide important insights into the role of host cell impurities on the performance of sterile filtration processes for therapeutic viruses.

Aerosol delivery, but not intramuscular injection, of adenovirus-vectored tuberculosis vaccine induces respiratory-mucosal immunity in humans.

BackgroundAdenovirus-vectored (Ad-vectored) vaccines are typically administered via i.m. injection to humans and are incapable of inducing respiratory mucosal immunity. However, aerosol delivery of Ad-vectored vaccines remains poorly characterized, and its ability to induce mucosal immunity in humans is unknown. This phase Ib trial evaluated the safety and immunogenicity of human serotype-5 Ad-vectored tuberculosis (TB) vaccine (AdHu5Ag85A) delivered to humans via inhaled aerosol or i.m. injection.MethodsThirty-one healthy, previously BCG-vaccinated adults were enrolled. AdHu5Ag85A was administered by single-dose aerosol using Aeroneb Solo Nebulizer or by i.m. injection. The study consisted of the low-dose (LD) aerosol, high-dose (HD) aerosol, and i.m. groups. The adverse events were assessed at various times after vaccination. Immunogenicity data were collected from the peripheral blood and bronchoalveolar lavage samples at baseline, as well as at select time points after vaccination.ResultsThe nebulized aerosol droplets were < 5.39 µm in size. Both LD and HD of AdHu5Ag85A administered by aerosol inhalation and i.m. injection were safe and well tolerated. Both aerosol doses, particularly LD, but not i.m., vaccination markedly induced airway tissue-resident memory CD4+ and CD8+ T cells of polyfunctionality. While as expected, i.m. vaccination induced Ag85A-specific T cell responses in the blood, the LD aerosol vaccination also elicited such T cells in the blood. Furthermore, the LD aerosol vaccination induced persisting transcriptional changes in alveolar macrophages.ConclusionInhaled aerosol delivery of Ad-vectored vaccine is a safe and superior way to elicit respiratory mucosal immunity. This study warrants further development of aerosol vaccine strategies against respiratory pathogens, including TB and COVID-19.Trial registrationClinicalTrial.gov, NCT02337270.FundingThe Canadian Institutes for Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada funded this work.

Invasive Myceliophthora thermophila infection mimicking invasive aspergillosis in a neutropenic patient: a new cause of cross-reactivity with the Aspergillus galactomannan serum antigen assay.

Myceliophthora thermophila is a thermophilic mould widely found in the environment but rarely responsible for human infections. We describe a case of invasive Myceliophthora thermophila infection mimicking invasive aspergillosis in a neutropenic patient with haematological malignancy. Cross-reactivity with Aspergillus galactomannan assay (GM) was demonstrated by repeated positive results and confirmed by cross-reaction between the fungal isolate and the GM assay. The patient was successfully treated with voriconazole. Potential GM cross-reactivity must be considered in future studies including patients categorized as having probable invasive aspergillosis using the GM as the only mycological criterion.

Disseminated mycosis in veiled chameleons (Chamaeleo calyptratus) caused by Chamaeleomyces granulomatis, a new fungus related to Paecilomyces viridis.

An outbreak of disseminated granulomatous disease occurred in a group of veiled chameleons (Chamaeleo calyptratus) in a zoo collection. An adult female and six offspring developed large granulomas in multiple organs and were euthanized. At necropsy, roughly spherical yellow-to-white nodules 1 to 3 mm in diameter were grossly visible in the liver and other organs. Histopathology revealed fungal elements that were spherical to ovoid in shape, fragments of slender to irregularly swollen hyphae, and occasional conidia produced on phialides. Fungal isolates were initially suspected on the basis of morphology results to represent Paecilomyces viridis, a species known only from one outbreak of fatal mycosis in carpet chameleons (Furcifer lateralis). Data obtained from morphological studies and from phylogenetic analyses of nuclear ribosomal rRNA (rDNA) sequence data revealed the Danish chameleon isolates to be a related undescribed anamorphic species within the family Clavicipitaceae that includes many insect pathogens. Chamaeleomyces granulomatis gen. et sp. nov. is given as the name for the newly described fungus, and P. viridis is transferred to the new genus as Chamaeleomyces viridis comb. nov. Chamaeleomyces species are distinguished by having basally swollen phialides tapering to a narrow neck, conidia in fragile chains, and pale green to greenish-gray colonies. Both species are dimorphic, producing a transitory yeast stage characterized by ovoid-to-subglobose or subcylindrical yeast-like cells. Chamaeleomyces species appear to be rare but aggressive pathogens of chameleons.

Phialosimplex, a new anamorphic genus associated with infections in dogs and having phylogenetic affinity to the Trichocomaceae.

Anamorphic members of the ascomycete family Trichocomaceae including Aspergillus, Penicillium, Paecilomyces, Geosmithia and Sagenomella have been reported from infections in canines. Six clinical isolates (five associated with infections in canines and one from a human source) demonstrated simple phialides producing conidia in long chains and were investigated for their potential relationship to Sagenomella chlamydospora, a known agent of canine disseminated mycosis. Phylogenetic analyses of internal transcribed spacer (ITS) and small subunit (SSU) region sequences revealed that all of the canine-associated isolates were distinct from Sagenomella species. The new anamorphic genus and species Phialosimplex caninus is described to accommodate the clinical isolates. Sagenomella chlamydospora and Sagenomella sclerotialis are transferred to the new genus as Phialosimplex chlamydosporus comb. nov. and Phialosimplex sclerotialis comb. nov.

Xylogone ganodermophthora sp. nov., an ascomycetous pathogen causing yellow rot on cultivated mushroom Ganoderma lucidum in Korea.

Yellow rot, caused by an ascomycetous fungus having a distinctive arthroconidial anamorph, is the most destructive disease of cultivated Ganoderma lucidum in Korea, but the identity of the yellow rot pathogen (YRP) remains uncertain. Isolates have been identified as Xylogone sphaerospora (with putative anamorph Sporendonema purpurascens) or as Arthrographis cuboidea. Therefore we used morphological features, pathogenicity tests and phylogenetic analyses of DNA sequences from the nuclear ribosomal genes, including partial small subunit and internal transcribed spacer regions, and from the gene encoding RNA polymerase second largest subunit to evaluate the relationship between YRP isolates and these species. YRP isolates formed a distinct subgroup within a clade that included X. sphaerospora, A. cuboidea and Scytalidium lignicola, the type species of Scytalidium, but the disposition of the clade within the Leotiomycetes was uncertain. We describe Xylogone ganodermophthora sp. nov. and Scytalidium ganodermophthorum sp. nov. for the teleomorph and anamorph of YRP respectively. Arthrographis cuboidea is reclassified as Scytalidium cuboideum comb. nov., and the anamorph of X. sphaerospora is named Scytalidium sphaerosporum sp. nov. In pathogenicity tests only X. ganodermophthora caused disease in Ganoderma lucidum. Amplified fragment length polymorphism analyses showed that X. ganodermophthora populations from diseased fruiting bodies or from oak wood in Korea consisted of two clonal groups.