National Research Institute

Campylobacter jejuni is a leading pathogen causing gastroenteritis in humans. Invasive C. jejuni strains crossing the gut-blood barrier can cause bacteremia. Despite its microaerophilicity (preferring <5% O2 atmosphere), hyper-aerotolerant (HAT) C. jejuni has recently been isolated from intestines in poultry and humans. Elevated aerotolerance may enhance C. jejuni survival in the ecosystem, thereby promoting its transmission. However, invasive HAT isolates of C. jejuni have not been reported in bacteremia in humans. A C. jejuni strain (CNH-HAT-1) was isolated from a 7% CO2 (~16% O2) atmosphere subculture of an aerobic blood culture from a 5-year-old boy with B-cell acute lymphoblastic leukemia presenting with bloody stool, fever, and neutropenia. The organism identification was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16S-rRNA sequencing. HAT phenotype of this isolate was confirmed by aerotolerance testing. Belonging to the ST-21 clonal complex, CNH-HAT-1 is phylogenetically close to recently reported invasive C. jejuni sheep abortion strains and some bacteremia-associated strains. CNH-HAT-1 possesses a CJIE-1 prophage element, a functional enterobactin receptor CfrB, and G250A mutation in the major outer-membrane protein PorA, which likely contributes to C. jejuni invasiveness. CNH-HAT-1 is resistant to penicillins/cephalosporins but susceptible to aminoglycosides, fluoroquinolones, and macrolides. The patient improved with intravenous gentamicin, followed by enteral therapy with azithromycin. We identified a novel invasive bloodstream C. jejuni strain proven in vitro to be HAT, shedding light on key genomic features linking with its pathogenesis. Given its enhanced survival of HAT C. jejuni in ecosystem, a clinical microbiology lab should be vigilant with detecting them from blood cultures.

Background:Variants in the mitochondrial genome (mtDNA) cause a diverse collection of mitochondrial diseases and have extensive phenotypic overlap with Mendelian diseases encoded on the nuclear genome. The mtDNA is often not specifically evaluated in patients with suspected Mendelian disease, resulting in overlooked diagnostic variants.Methods:Using dedicated pipelines to address the technical challenges posed by the mtDNA - circular genome, variant heteroplasmy, and nuclear misalignment - single nucleotide variants, small indels, and large mtDNA deletions were called from exome and genome sequencing data, in addition to RNA-sequencing when available. A cohort of 6,660 rare disease families were analyzed (5,625 genetically undiagnosed, 84%) from the Genomics Research to Elucidate the Genetics of Rare diseases (GREGoR) Consortium as well as other rare disease cohorts.Results:Diagnostic mtDNA variants were identified in 10 previously genetically undiagnosed families (one large deletion, eight reported pathogenic variants, one novel pathogenic variant). In one additional undiagnosed proband, the detection of >900 heteroplasmic variants provided functional evidence of pathogenicity to a novel de novo variant in the nuclear gene POLG (DNA polymerase gamma), responsible for mtDNA replication and repair.Conclusion:mtDNA variant calling from data generated by exome and genome sequencing for nuclear variant analysis resulted in a genetic diagnosis or detection of a candidate variant for 0.4% of undiagnosed families affected by a broad range of rare diseases.

Chronic red blood cell (RBC) transfusion is an established therapy to prevent stroke in patients with sickle cell anemia (SCA). It is unclear if adding daily hydroxyurea treatment to chronic transfusion is beneficial. We conducted a phase 2 clinical trial (NCT03644953) investigating the addition of dose-escalated hydroxyurea to chronic transfusion for patients with SCA receiving simple chronic transfusion for stroke prevention. Simple chronic transfusion therapy was administered as per the same protocol before and after hydroxyurea treatment in which the volume transfused was dependent on the pretransfusion hemoglobin (Hb). A total of 14 participants enrolled with nine completing one year of combination hydroxyurea and transfusion (HAT) therapy after reaching hydroxyurea target dose. No participant who discontinued the study prematurely had a serious adverse event attributed to HAT. Among the nine participants who completed the study, eight participants achieved a reduction in RBC transfusion volume with a median reduction of -19.4 mL/kg/year (interquartile range -31.8, -2.8 mL/kg/year), p = .02, when comparing pre- and post-HAT time periods. With the addition of hydroxyurea participants had a significant increase in pretransfusion Hb S% but this was balanced by an increased Hb F% and decreased lactate dehydrogenase. One participant developed a pretransfusion Hb >11 g/dL and Hb S > 45% that required holding hydroxyurea and changing to partial manual exchange transfusions. No patient had evidence of cerebrovascular disease progression. Hydroxyurea added to chronic transfusion therapy for patients with SCA is feasible and decreases RBC transfusion volume requirements.

Background: Myofibroblasts (MYFs) are generally considered the principal culprits in excessive extracellular matrix deposition and scar formation in the pathogenesis of lung fibrosis. Lipofibroblasts (LIFs), on the other hand, are defined by their lipid-storing capacity and are predominantly found in the alveolar regions of the lung. They have been proposed to play a protective role in lung fibrosis. We previously reported that a LIF to MYF reversible differentiation switch occurred during fibrosis formation and resolution. In this study, we tested whether WI-38 cells, a human embryonic lung fibroblast cell line, could be used to study fibroblast differentiation towards the LIF or MYF phenotype and whether this could be relevant for idiopathic pulmonary fibrosis (IPF). Methods: Using WI-38 cells, Fibroblast (FIB) to MYF differentiation was triggered using TGF-β1 treatment and FIB to LIF differentiation using Metformin treatment. We also analyzed the MYF to LIF and LIF to MYF differentiation by pre-treating the WI-38 cells with TGF-β1 or Metformin respectively. We used IF, qPCR and bulk RNA-Seq to analyze the phenotypic and transcriptomic changes in the cells. We correlated our in vitro transcriptome data from WI-38 cells (obtained via bulk RNA sequencing) with the transcriptomic signature of LIFs and MYFs derived from the IPF cell atlas as well as with our own single-cell transcriptomic data from IPF patients-derived lung fibroblasts (LF-IPF) cultured in vitro. We also carried out alveolosphere assays to evaluate the ability of the proposed LIF and MYF cells to support the growth of alveolar epithelial type 2 cells. Results: WI-38 cells and LF-IPF display similar phenotypical and gene expression responses to TGF-β1 and Metformin treatment. Bulk RNA-Seq analysis of WI-38 cells and LF-IPF treated with TGF-β1, or Metformin indicate similar transcriptomic changes. We also show the partial conservation of the LIF and MYF signature extracted from the Habermann et al. scRNA-seq dataset in WI-38 cells treated with Metformin or TGF-β1, respectively. Alveolosphere assays indicate that LIFs enhance organoid growth, while MYFs inhibit organoid growth. Finally, we provide evidence supporting the MYF to LIF and LIF to MYF reversible switch using WI-38 cells. Conclusions: WI-38 cells represent a versatile and reliable model to study the intricate dynamics of fibroblast differentiation towards the MYF or LIF phenotype associated with lung fibrosis formation and resolution, providing valuable insights to drive future research.

To evaluate the role of focal cortical dysplasia co-localization to cortical functional networks in the development of pharmacoresistance. One hundred thirty-six focal cortical dysplasia patients with 3.0 T or 1.5 T MRI were identified from clinical databases at Children's National Hospital. Clinico-radio-pathologic factors and network co-localization were determined. Using binomial logistic regression, limbic network co-localization (odds ratio 4.164 95% confidence interval 1.02-17.08, p = 0.048), and focal to bilateral tonic-clonic seizures (4.82, 1.30-18.03, p = 0.019) predicted pharmacoresistance. These findings provide clinicians with markers to identify patients with focal cortical dysplasia-related epilepsy at high risk of developing pharmacoresistance and should facilitate earlier epilepsy surgical evaluation.

Abstract Nematodes represent different trophic groups in the soil. They therefore play an important role in the soil environment: they regulate populations of other soil organisms, mineralize nutrients into plant-available forms, provide a source of food for other soil organisms and feed on pathogenic organisms and plant pests. In contrast, plant-parasitic nematodes are important pests of crops. Nanoparticles are increasingly being used in agriculture and other production sectors. They are present in the soil, not necessarily in trace amounts, and can affect a variety of soil organisms, including nematodes. Under laboratory conditions, the effects of silver (Ag), gold (Au) and platinum (Pt) nanoparticles on the biological activity of selected nematode species from two trophic groups: entomopathogenic nematodes (Heterorhabditis bacteriophora and Steinernema feltiae) and plant parasitic nematodes (Xiphinema diversicaudatum, Ditylenchus dipsaci, Heterodera schachtii) were studied. The plant-parasitic nematodes tested responded differently to nanoparticle solutions. The greatest sensitivity to Ag nanoparticles was observed in D. dipsaci. The best effect of Au nanoparticles was observed in H. schachtii. Lead caused a positive response in D. dipsaci, in contrast to H. schachtii, compared to the control. In contrast, X. diversicaudatum and H. baltica were not affected by Pt nanoparticles. Despite similar concentrations silver nanoparticles, compared to Au and Pt nanoparticles, had the strongest toxic effects on the infective juveniles of both EPN nematode species tested. However, during the migration of a new generation of EPN infective juveniles from a dead insect, the results were inconclusive. For H. bacteriophora, the number of IJs nematodes migrating outside the cadaver after contact with AgNP and PtNP was higher compared to the control and other nanoparticles. The results encourage field research into increasing the efficacy of entomopathogenic nematode biopreparations using nanoparticles and reducing the harmfulness of plant parasitic nematodes in crops.

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Cystic fibrosis (CF) is an important monogenic disease that affects more than 70 000 people worldwide. Defects of the CF transmembrane conductance regulator gene lead to dehydrated viscous secretions that result in chronic bacterial colonization. This leads to frequent recurrent lung infections called pulmonary exacerbations, lung inflammation, and resulting structural lung damage called bronchiectasis. Pseudomonas aeruginosa in particular is a common pathogen in persons with CF associated with increased pulmonary exacerbations, long-term lung function decline, and reduced survival. In addition, P. aeruginosa commonly develops antibiotic resistance and forms biofilms, making it difficult to treat. Here, we report the details of two patients with CF with pan-drug-resistant P. aeruginosa who were treated with a novel therapeutic strategy, bacteriophages. These cases highlight the need for further research and development of this treatment modality, including pediatric clinical trials.