Immunogenicity of trimeric autotransporter adhesins and their potential as vaccine targets

The current problem of increasing antibiotic resistance and the resurgence of numerous infections indicate the need for novel vaccination strategies more than ever. In vaccine development, the search for and the selection of adequate vaccine antigens is the first important step. In recent years, bacterial outer membrane proteins have become of major interest, as they are the main proteins interacting with the extracellular environment. Trimeric autotransporter adhesins (TAAs) are important virulence factors in many Gram-negative bacteria, are localised on the bacterial surface, and mediate the first adherence to host cells in the course of infection. One example is the Neisseria adhesin A (NadA), which is currently used as a subunit in a licensed vaccine against Neisseria meningitidis. Other TAAs that seem promising vaccine candidates are the Acinetobacter trimeric autotransporter (Ata), the Haemophilus influenzae adhesin (Hia), and TAAs of the genus Bartonella. Here, we review the suitability of various TAAs as vaccine candidates.Peer reviewe

Nursing considerations to complement the Surviving Sepsis Campaign guidelines

Objectives: To provide a series of recommendations based on the best available evidence to guide clinicians providing nursing care to patients with severe sepsis. Design: Modified Delphi method involving international experts and key individuals in subgroup work and electronic-based discussion among the entire group to achieve consensus. Methods: We used the Surviving Sepsis Campaign guidelines as a framework to inform the structure and content of these guidelines. We used the Grades of Recommendation, Assessment, Development, and Evaluation (GRADE) system to rate the quality of evidence from high (A) to very low (D) and to determine the strength of recommendations, with grade 1 indicating clear benefit in the septic population and grade 2 indicating less confidence in the benefits in the septic population. In areas without complete agreement between all authors, a process of electronic discussion of all evidence was undertaken until consensus was reached. This process was conducted independently of any funding. Results: Sixty-three recommendations relating to the nursing care of severe sepsis patients are made. Prevention recommendations relate to education, accountability, surveillance of nosocomial infections, hand hygiene, and prevention of respiratory, central line-related, surgical site, and urinary tract infections, whereas infection management recommendations related to both control of the infection source and transmission-based precautions. Recommendations related to initial resuscitation include improved recognition of the deteriorating patient, diagnosis of severe sepsis, seeking further assistance, and initiating early resuscitation measures. Important elements of hemodynamic support relate to improving both tissue oxygenation and macrocirculation. Recommendations related to supportive nursing care incorporate aspects of nutrition, mouth and eye care, and pressure ulcer prevention and management. Pediatric recommendations relate to the use of antibiotics, steroids, vasopressors and inotropes, fluid resuscitation, sedation and analgesia, and the role of therapeutic end points. Conclusion: Consensus was reached regarding many aspects of nursing care of the severe sepsis patient. Despite this, there is an urgent need for further evidence to better inform this area of critical care

A multimodal cell census and atlas of the mammalian primary motor cortex

ABSTRACT We report the generation of a multimodal cell census and atlas of the mammalian primary motor cortex (MOp or M1) as the initial product of the BRAIN Initiative Cell Census Network (BICCN). This was achieved by coordinated large-scale analyses of single-cell transcriptomes, chromatin accessibility, DNA methylomes, spatially resolved single-cell transcriptomes, morphological and electrophysiological properties, and cellular resolution input-output mapping, integrated through cross-modal computational analysis. Together, our results advance the collective knowledge and understanding of brain cell type organization: First, our study reveals a unified molecular genetic landscape of cortical cell types that congruently integrates their transcriptome, open chromatin and DNA methylation maps. Second, cross-species analysis achieves a unified taxonomy of transcriptomic types and their hierarchical organization that are conserved from mouse to marmoset and human. Third, cross-modal analysis provides compelling evidence for the epigenomic, transcriptomic, and gene regulatory basis of neuronal phenotypes such as their physiological and anatomical properties, demonstrating the biological validity and genomic underpinning of neuron types and subtypes. Fourth, in situ single-cell transcriptomics provides a spatially-resolved cell type atlas of the motor cortex. Fifth, integrated transcriptomic, epigenomic and anatomical analyses reveal the correspondence between neural circuits and transcriptomic cell types. We further present an extensive genetic toolset for targeting and fate mapping glutamatergic projection neuron types toward linking their developmental trajectory to their circuit function. Together, our results establish a unified and mechanistic framework of neuronal cell type organization that integrates multi-layered molecular genetic and spatial information with multi-faceted phenotypic properties

Identifizierung und funktionelle Analyse von Pathogenitätsfaktoren in Bartonella bacilliformis

Die Carrión-Krankheit ist eine durch Vektoren übertragene vernachlässigte Tropenkrankheit (neglected tropical disease), die in den südamerikanischen Andentälern auf einer Höhe von 600 3.200 m über dem Meeresspiegel vor allem in Peru, aber auch in Ecuador und Kolumbien endemisch ist. Der Erreger dieser Infektionskrankheit ist Bartonella bacilliformis, ein strikt humanpathogenes, Gram-negatives, fakultativ intrazelluläres Stäbchen der Klasse der Alphaproteobakterien. In der akuten Krankheitsphase, die als "Oroya-Fieber" bezeichnet wird, infizieren die Erreger Erythrozyten und verursachen eine schwere akute hämolytische Anämie, hohes Fieber sowie eine ausgeprägte Immunsuppression. Für diese Phase wurden Sterblichkeitsraten von bis zu 88% beschrieben. Dem Oroya-Fieber folgt meist eine chronische Infektion der vaskulären Endothelzellen, bei der durch vaskulo-endotheliale Proliferationen noduläre, Hämangiom-ähnliche, kutane Gefäßläsionen, die als "Verruga peruana" bezeichnet werden, entstehen. Diese beiden Phasen treten in der Regel nacheinander, manchmal aber auch unabhängig voneinander auf. Die Übertragung auf dem Menschen erfolgt durch den Biss infizierter Sandmücken (Lutzomyia spp.), die in den hochgelegenen Regionen der Anden vorkommen. Klimatische Veränderungen führen jedoch zur Expansion des Vektors auf angrenzende Regionen und begünstigen damit die Ausbreitung von B. bacilliformis-Infektionen. In der Erforschung der Carrión-Krankheit besteht ein erheblicher Wissensmangel zu zahlreichen Aspekten (z. B. Epidemiologie, Infektionsbiologie, Diagnostik, Therapie), wodurch die Entwicklung von potenziellen Diagnostika, Therapeutika oder Vakzinen verhindert wird. Auch wenn frühere Studien zum Ziel hatten, immundominante Proteine für die Entwicklung serodiagnostischer Verfahren und Impfstoffe zu identifizieren, ist bislang kein validierter serologischer Test bzw. ein Impfstoff verfügbar. Daher sollte im ersten Teil dieser Arbeit ein serologischer Test zum Nachweis von anti-B. bacilliformis-Antikörpern entwickelt werden. Hierzu wurde ein Ansatz aus reverser Vakzinologie in Kombination mit einer Analyse heterologer genomischer Expressionsbibliotheken verfolgt, um geeignete immundominante Proteine zu identifizieren. Insgesamt wurden 21 potenziell immundominante Proteine identifiziert, rekombinant produziert, und auf ihre Reaktivität mit B. bacilliformis Patientenseren mittels Immunoblotting analysiert. Von den 21 Antigenkandidaten erwiesen sich 14 als immunreaktiv, die anschließend in einer Lineblot-Analyse mit 26 Serumproben von peruanischen B. bacilliformis Patienten und 96 Serumproben von gesunden deutschen Blutspendern ohne Reisevorgeschichte in Südamerika auf ihren potenziellen Nutzen für serologische Test untersucht wurden. Drei Antigene (Porin-B, Autotransporter-E und hypothetisches Protein-B) erwiesen sich für die Entwicklung eines diagnostischen ELISA als geeignet und wurden in zwei verschiedenen Antigenkombinationen (ELISA 1: Porin-B, Autotransporter-E, ELISA 2: Porin-B, Autotransporter-E, hypothetisches Protein B) verwendet. Um die Leistungsfähigkeit des B. bacilliformis ELISA zu bewerten, wurde eine Receiver-Operating-Characteristic-Analyse durchgeführt. Für die Kombination aus Porin-B und Autotransporter-E lag die Sensitivität des Tests bei 80,8% und die Spezifität bei 94,8%, wohingegen die Kombination aus Porin-B, Autotransporter-E und dem hypothetischem Protein-B in einer Sensitivität von 76,9% und einer Spezifität von 93,8% resultierte. Dieser neu entwickelte ELISA könnte ein nützliches serodiagnostisches Instrument für künftige epidemiologische Studien über B. bacilliformis in endemischen Gebieten darstellen. Darüber hinaus könnten die hier identifizierten immundominanten Antigene eine erste Grundlage für die zukünftige Entwicklung von Impfstoffen für die Prävention Carrión-Krankheit bilden. Erythrozyten-Invasion und Hämolyse sind wahrscheinlich die wichtigsten Schritte in der Pathogenese der Carrión-Krankheit und verantwortlich für die hohe Sterblichkeitsrate beim Menschen. Genaue mechanistische Kenntnis dieses Prozesses sind entscheidend für die Entwicklung therapeutischer Arzneimittel. Im zweiten Teil dieser Arbeit sollten die in der Hämolyse involvierten Pathogenitätsfaktoren von B. bacilliformis identifiziert werden. Hierzu wurde eine Tn5-Transposonmutagenese in den beiden B. bacilliformis Stämmen KC583 und KC584 durchgeführt. Ein screening nach Hämolyse-defizienten Mutanten führte zur Identifizierung von zwei Pathogenitätsfaktoren: einem Porin (Porin-A) und einer α/β-Hydrolase. Ihre vermutete Funktion im Prozess der Hämolyse wurde durch eine zielgerichtete markerlose Deletion sowie durch genetische Komplementationen in vitro funktionell bestätigt. Dabei zeigte sich, dass Porin-A und die α/β Hydrolase jeweils essenzielle Faktoren für die Hämolyse darstellen. Durch eine in silico-Charakterisierung konnte konservierte biologische Funktionen identifiziert sowie die dreidimensionale Struktur der Proteine vorhergesagt werden. Diese Versuche bilden eine experimentelle Basis, um die Rolle von Porin-A und der α/β-Hydrolase näher untersuchen zu können und um potenzielle (Porin-A und α/β-Hydrolase) Inhibitoren mit anti-hämolytischer und damit therapeutische Wirkung testen zu können.Carrion's disease is a vector-borne neglected tropical disease that is endemic to the South American Andes with Peru as the most affected country. The causative agent of this infectious disease is Bartonella bacilliformis, a Gram-negative, facultative intracellular alphaproteobacterium. In the acute phase of the disease, known as "Oroya fever", the pathogen infects erythrocytes and causes a severe acute hemolytic anemia, high fever, and immunosuppression. For this phase mortality rates of up to 88% have been described. Oroya fever is often followed by a chronic infection of the vascular endothelium, in which the bacteria stimulate vascular-endothelial proliferation which leads to the formation of nodular, hemangioma-like cutaneous lesions known as "verruga peruana". Humans are the only known reservoirs and the transmission occurs through the bite of infected sandflies (Lutzomyia spp.), which are found in the high-altitude regions of the Andes. Climatic changes lead to the expansion of the vector to neighboring regions and thus favor the spread of B. bacilliformis infections. There is a significant lack of knowledge on many aspects of Carrion's disease research, which prevents the development of potential diagnostics, therapeutics, and vaccines. Although previous studies have aimed to identify antigens, no validated serological test or vaccine is yet available. Therefore, the first part of this work aimed to develop a serological test for the detection of anti-B. bacilliformis antibodies. For this purpose, a combination of reverse vaccinology and heterologous genomic expression libraries was used to identify suitable immunodominant proteins. A total of 21 potentially immunodominant proteins were identified, recombinantly produced and analyzed for their reactivity with B. bacilliformis patient sera by using immunoblotting. Of the 21 antigen candidates, 14 were found to be immunoreactive and were further analyzed for their serodiagnostic utility by using a line blot analysis with 26 serum samples from Peruvian B. bacilliformis patients and 96 serum samples from healthy German blood donors with no history of travel to South America. Three antigens (porin-B, autotransporter-E and hypothetical protein-B) proved to be suitable for the development of a diagnostic ELISA and were tested in two different antigen combinations (ELISA 1: porin-B, autotransporter-E, ELISA 2: porin-B, autotransporter-E, hypothetical protein-B). The performance of the B. bacilliformis ELISA was evaluated by a receiver operating characteristic analysis. The ELISA using the antigen combination of porin-B and autotransporter-E, achieved a sensitivity of 80.8% and a specificity of 94.8%, whereas the combination of porin-B, autotransporter-E and hypothetical protein-B resulted in a sensitivity of 76.9% and a specificity of 93.8%. This newly developed ELISA could be a useful serodiagnostic tool for future epidemiologic studies of B. bacilliformis in endemic areas. In addition, the immunodominant antigens identified in this work could provide an initial basis for the future development of protective vaccines against Carrion's disease. Erythrocyte invasion and hemolysis are probably the most important steps in the pathogenesis of Carrion's disease and are responsible for the high mortality rate in humans. Detailed knowledge of these processes is crucial for the development of therapeutic drugs. In the second part of this work, hemolysis-related pathogenicity factors of B. bacilliformis were identified. For this purpose, a Tn5 transposon mutagenesis was performed in B. bacilliformis and screening for non-hemolytic mutants led to the identification of two pathogenicity factors: a porin (porin-A) and an α/β-hydrolase. Their presumed function in the hemolysis process was functionally confirmed in vitro by targeted markerless deletion and complementation of both genes. The results showed that porin-A and the α/β hydrolase are each essential factors for the hemolytic activity. These experiments provide an experimental basis to further investigate the role of porin-A and α/β-hydrolase and to test potential (porin-A and α/β-hydrolase) inhibitors for the development of new therapeutic strategies

Carrion’s disease: more than a neglected disease

Abstract Infections with Bartonella bacilliformis result in Carrion’s disease in humans. In the first phase of infection, the pathogen causes a hemolytic fever (“Oroya fever”) with case-fatality rates as high as ~90% in untreated patients, followed by a chronical phase resulting in angiogenic skin lesions (“verruga peruana”). Bartonella bacilliformis is endemic to South American Andean valleys and is transmitted via sand flies (Lutzomyia spp.). Humans are the only known reservoir for this old disease and therefore no animal infection model is available. In the present review, we provide the current knowledge on B. bacilliformis and its pathogenicity factors, vectors, possible unknown reservoirs, established and potential infection models and immunological aspects of the disease

Action of GP 47779, the active metabolite of oxcarbazepine, on the corticostriatal system. I. Modulation of corticostriatal synaptic transmission

Oxcarbazepine (OCBZ) is the keto-analogue of carbamazepine (CBZ). In humans, OCBZ is rapidly and almost completely metabolized to 10, 11-dihydro-10-hydroxy-CBZ (GP 47779), the main metabolite responsible for the drug's antiepileptic activity. The corticostriatal pathway is involved in the propagation of epileptic discharges. We characterized the electrophysiological effects of GP 47779 on striatal neurons by making intracellular recordings from corticostriatal slices. GP 47779 (3-100 microM) produced a dose-dependent inhibition of glutamatergic excitatory postsynaptic potentials (EPSPs). This effect was not coupled either with changes of the membrane potential of these cells or with alterations of their postsynaptic sensitivity to excitatory amino acids (EAA) suggesting a presynaptic site of action. GP 47779 reduced the current-evoked firing discharge only at concentrations > 100 microM. GP 47779 did not affect the presynaptic inhibitory action of adenosine, showing that presynaptic adenosine receptors were not implicated in the GP 47779-mediated reduction of corticostriatal EPSPs. Our data indicate that GP 47779 apparently acts directly on corticostriatal terminals to reduce the release of EAA, probably by inhibiting high-voltage-activated (HVA) calcium (Ca2+) currents (described in the accompanying article). The inhibitory action of GP 47779 on corticostriatal transmission may contribute to the antiepileptic effects of this drug