Novel Immunomodulatory Flagellin-Like Protein FlaC in Campylobacter jejuni and Other Campylobacterales

The human diarrheal pathogens Campylobacter jejuni and Campylobacter coli interfere with host innate immune signaling by different means, and their flagellins, FlaA and FlaB, have a low intrinsic property to activate the innate immune receptor Toll-like receptor 5 (TLR5). We have investigated here the hypothesis that the unusual secreted, flagellin-like molecule FlaC present in C. jejuni, C. coli, and other Campylobacterales might activate cells via TLR5 and interact with TLR5. FlaC shows striking sequence identity in its D1 domains to TLR5-activating flagellins of other bacteria, such as Salmonella, but not to nonstimulating Campylobacter flagellins. We overexpressed and purified FlaC and tested its immunostimulatory properties on cells of human and chicken origin. Treatment of cells with highly purified FlaC resulted in p38 activation. FlaC directly interacted with TLR5. Preincubation with FlaC decreased the responsiveness of chicken and human macrophage-like cells toward the bacterial TLR4 agonist lipopolysaccharide (LPS), suggesting that FlaC mediates cross-tolerance. C. jejuni flaC mutants induced an increase of cell responses in comparison to those of the wild type, which was suppressed by genetic complementation. Supplementing excess purified FlaC likewise reduced the cellular response to C. jejuni. In vivo, the administration of ultrapure FlaC led to a decrease in cecal interleukin 1β (IL-1β) expression and a significant change of the cecal microbiota in chickens. We propose that Campylobacter spp. have evolved a novel type of secreted immunostimulatory flagellin-like effector in order to specifically modulate host responses, for example toward other pattern recognition receptor (PRR) ligands, such as LPS. IMPORTANCE Flagellins not only are important for bacterial motility but are major bacterial proteins that can modulate host responses via Toll-like receptor 5 (TLR5) or other pattern recognition receptors. Campylobacterales colonizing the intestinal tracts of different host species harbor a gene coding for an unusual flagellin, FlaC, that is not involved in motility but is secreted and possesses a chimeric amino acid sequence composed of TLR5-activating and non-TLR5-activating flagellin sequences. Campylobacter jejuni FlaC activates cells to increase in cytokine expression in chicken and human cells, promotes cross-tolerance to TLR4 ligands, and alters chicken cecal microbiota. We propose that FlaC is a secreted effector flagellin that has specifically evolved to modulate the immune response in the intestinal tract in the presence of the resident microbiota and may contribute to bacterial persistence. The results also strengthen the role of the flagellar type III apparatus as a functional secretion system for bacterial effector proteins

Gibt es Hinweise auf eine Beteiligung von Sarcocystis calchasi an Meningoenzephalitiden unbekannter Genese bei Säugetieren? Eine retrospektive Studie

Sarcocystis calchasi is a novel intracellular protozoan parasite belonging to the phy-lum Apicomplexa with an obligatory two-host, predator-prey life cycle. The northern goshawk (Accipiter gentilis) is its definitive host and the domestic pigeon (Columba livia f. domestica) its intermediate host. It has been identified as the causative agent of Pigeon Protozoal En-cephalitis (PPE) during an outbreak in Berlin in the period between 2006 and 2008. PPE is an ongoing threat as new cases are continuously diagnosed in pigeons in the Berlin area. Since many Sarcocystis spp. are polyxenous, being capable of infecting more than one species, and birds as well as mammals may usually serve as their intermediate hosts, a retrospective study was conducted to determine whether Sarcocystis calchasi may be in-volved in cases of meningoencephalitis of unknown origin (MUO) in mammals. Formalin fixed paraffin embedded (FFPE) samples of 143 brains with MUO of different mammalian species (dog, cat, pig, cattle, sheep, guinea pig, horse, goat, mouse, raccoon, ferret, hamster, mink, mane wolf) from the time period between 1989 and 2012 were reex-amined histologically using H&E; stain. DNA was isolated from FFPE material and screened by polymerase chain reaction (PCR) with primers specific for the 18S rRNA and the Internal Transcribed Spacer region 1 (ITS1) to detect Sarcocystis calchasi or other apicomplexan parasites, respectively. In all samples the diagnosis of non-suppurative (lymphoplasmacytic and / or granu-lomatous) meningoencephalitis was histologically confirmed but no parasitic structures were found in the histopathological investigation. DNA of Sarcocystis calchasi or other apicom- plexan parasites could not be detected in any of the samples. However due to formalin fixa-tion the DNA quality of the samples might not have been optimal in all cases, which is why the results should be interpreted carefully. Even though the prevalence of PPE in pigeons remains constant and Sarcocystis cal- chasi constitutes a persistent threat to pigeons in the Berlin area, no evidence was found here for a role of this parasite in MUO in mammalian species. Nevertheless, taking into con-sideration the methodical drawbacks of analyzing FFPE retrospective cases, a prospective study using new emerging cases with optimal DNA quality and / or immunohistochemical studies with specific antibodies would be advisable.Sarcocystis calchasi ist ein intrazellulärer, einzelliger Parasit, der zum Stamm Apicomplexa gehört. Der Parasit hat einen obligatorischen zwei-wirtigen Lebenszyklus. Der Habicht (Accipiter gentilis) ist der Endwirt und die Taube (Columba livia f. domestica) ist der Zwischenwirt. Sarcocystis calchasi ist als Erreger der Pigeon Protozoal Enzephalitis (PPE) während eines Ausbruchs dieser Krankheit in Berlin in der Zeit zwischen 2006 und 2008 identifiziert worden. Seit 2008 konnte eine konstante Inzidenz der Krankheit beobachtet werden. Diese Beobachtungen sprechen für eine dauerhafte Belastung der Taubenpopulation in dieser Region. Da viele Sarcocystis spp. mehrwirtig und in der Lage sind mehrere Tierarten zu infizieren, könnten sowohl Vögel als auch Säugetiere grundsätzlich als Zwischenwirte dienen. Deswegen wurde eine retrospektive Studie durchgeführt, um festzustellen, ob Sarcocystis calchasi in Fällen von Meningoenzephalitis unbekannter Genese (meningoencephalitis of unknown origin, MUO) bei Säugetieren beteiligt sein könnte. Formalinfixierte und in Paraffin eingebettete (FFPE) Proben von 143 Gehirnen mit MUO verschiedener Säugetierspezies (Hund, Katze, Schwein, Rind, Schaf, Meerschweinchen, Pferd, Ziege, Maus, Waschbär, Frettchen, Hamster, Nerz, Mähnenwolf) aus dem Zeitraum zwischen 1989 und 2012 wurden histologisch erneut untersucht. DNA wurde aus FFPE-Material isoliert und mittels Polymerase- Kettenreaktion (PCR) mit spezifischen Primern für die 18S rRNA und die Internal Transcribed Spacer-Region 1 (ITS1) untersucht, um Sarcocystis calchasi oder andere Apicomplexa nachzuweisen. In allen Proben wurde die Diagnose einer nicht-eitrigen (lymphoplasmazytären und / oder granulomatösen) Meningoenzephalitis bestätigt, jedoch konnten keine parasitären Strukturen in der histopathologischen Untersuchung festgestellt werden. DNA von Sarcocystis calchasi oder von anderen Apicomplexa konnte in keiner der Proben nachgewiesen werden. Alle Kontrollproben dagegen verliefen einwandfrei. Aufgrund Formalinfixierung könnte jedoch die DNA Qualität der Proben beeinträchtigt gewesen sein, weshalb die Ergebnisse vorsichtig interpretiert werden sollten. Auch wenn die Prävalenz der PPE konstant bleibt und Sarcocystis calchasi eine anhaltende Bedrohung für die Tauben im Berliner Raum darstellt, wurden keine Hinweise für eine Rolle dieses Parasiten bei MUO von Säugetieren festgestellt. Unter Berücksichtigung der methodischen Nachteile einer retrospektiven Analyse von FFPE-Proben wären jedoch eine prospektive Studie der neu auftretenden Fälle mit optimaler DNA-Qualität und / oder immunohistochemischen Untersuchungen mit spezifischen Antikörpern empfehlenswert

Mini Review: The Last Mile—Opportunities and Challenges for Machine Learning in Digital Toxicologic Pathology

The 2019 manuscript by the Special Interest Group on Digital Pathology and Image Analysis of the Society of Toxicologic pathology suggested that a synergism between artificial intelligence (AI) and machine learning (ML) technologies and digital toxicologic pathology would improve the daily workflow and future impact of toxicologic pathologists globally. Now 2 years later, the authors of this review consider whether, in their opinion, there is any evidence that supports that thesis. Specifically, we consider the opportunities and challenges for applying ML (the study of computer algorithms that are able to learn from example data and extrapolate the learned information to unseen data) algorithms in toxicologic pathology and how regulatory bodies are navigating this rapidly evolving field. Although we see similarities with the “Last Mile” metaphor, the weight of evidence suggests that toxicologic pathologists should approach ML with an equal dose of skepticism and enthusiasm. There are increasing opportunities for impact in our field that leave the authors cautiously excited and optimistic. Toxicologic pathologists have the opportunity to critically evaluate ML applications with a “call-to-arms” mentality. Why should we be late adopters? There is ample evidence to encourage engagement, growth, and leadership in this field

Detection of MCPG metabolites in horses with atypical myopathy.

Atypical myopathy (AM) in horses is caused by ingestion of seeds of the Acer species (Sapindaceae family). Methylenecyclopropylacetyl-CoA (MCPA-CoA), derived from hypoglycin A (HGA), is currently the only active toxin in Acer pseudoplatanus or Acer negundo seeds related to AM outbreaks. However, seeds or arils of various Sapindaceae (e.g., ackee, lychee, mamoncillo, longan fruit) also contain methylenecyclopropylglycine (MCPG), which is a structural analogue of HGA that can cause hypoglycaemic encephalopathy in humans. The active poison formed from MCPG is methylenecyclopropylformyl-CoA (MCPF-CoA). MCPF-CoA and MCPA-CoA strongly inhibit enzymes that participate in β-oxidation and energy production from fat. The aim of our study was to investigate if MCPG is involved in Acer seed poisoning in horses. MCPG, as well as glycine and carnitine conjugates (MCPF-glycine, MCPF-carnitine), were quantified using high-performance liquid chromatography-tandem mass spectrometry of serum and urine from horses that had ingested Acer pseudoplatanus seeds and developed typical AM symptoms. The results were compared to those of healthy control horses. For comparison, HGA and its glycine and carnitine derivatives were also measured. Additionally, to assess the degree of enzyme inhibition of β-oxidation, several acyl glycines and acyl carnitines were included in the analysis. In addition to HGA and the specific toxic metabolites (MCPA-carnitine and MCPA-glycine), MCPG, MCPF-glycine and MCPF-carnitine were detected in the serum and urine of affected horses. Strong inhibition of β-oxidation was demonstrated by elevated concentrations of all acyl glycines and carnitines, but the highest correlations were observed between MCPF-carnitine and isobutyryl-carnitine (r = 0.93) as well as between MCPA- (and MCPF-) glycine and valeryl-glycine with r = 0.96 (and r = 0.87). As shown here, for biochemical analysis of atypical myopathy of horses, it is necessary to take MCPG and the corresponding metabolites into consideration

Domain Adaptation-Based Deep Learning for Automated Tumor Cell (TC) Scoring and Survival Analysis on PD-L1 Stained Tissue Images.

We report the ability of two deep learning-based decision systems to stratify non-small cell lung cancer (NSCLC) patients treated with checkpoint inhibitor therapy into two distinct survival groups. Both systems analyze functional and morphological properties of epithelial regions in digital histopathology whole slide images stained with the SP263 PD-L1 antibody. The first system learns to replicate the pathologist assessment of the Tumor Cell (TC) score with a cut-point for positivity at 25% for patient stratification. The second system is free from assumptions related to TC scoring and directly learns patient stratification from the overall survival time and event information. Both systems are built on a novel unpaired domain adaptation deep learning solution for epithelial region segmentation. This approach significantly reduces the need for large pixel-precise manually annotated datasets while superseding serial sectioning or re-staining of slides to obtain ground truth by cytokeratin staining. The capacity of the first system to replicate the TC scoring by pathologists is evaluated on 703 unseen cases, with an addition of 97 cases from an independent cohort. Our results show Lin's concordance values of 0.93 and 0.96 against pathologist scoring, respectively. The ability of the first and second system to stratify anti-PD-L1 treated patients is evaluated on 151 clinical samples. Both systems show similar stratification powers (first system: HR = 0.539, p = 0.004 and second system: HR = 0.525, p = 0.003) compared to TC scoring by pathologists (HR = 0.574, p = 0.01)