Precision Cut Lung Slices (PCLS) have emerged as a sophisticated and physiologically relevant ex vivo model for studying the intricacies of lung diseases, including fibrosis, injury, repair, and host defense mechanisms. This innovative methodology presents a unique opportunity to bridge the gap between traditional in vitro cell cultures and in vivo animal models, offering researchers a more accurate representation of the intricate microenvironment of the lung. PCLS require the precise sectioning of lung tissue to maintain its structural and functional integrity. These thin slices serve as invaluable tools for various research endeavors, particularly in the realm of airway diseases. By providing a controlled microenvironment, precision-cut lung slices empower researchers to dissect and comprehend the multifaceted interactions and responses within lung tissue, thereby advancing our understanding of pulmonary pathophysiology. 

pdf/precision-cut-lung-slices-an-integrated-ex-vivo-model-for-1d2viwqdsk.pdf

Precision cut lung slices: an integrated ex vivo model for studying lung physiology, pharmacology, disease pathogenesis and drug discovery

Pneumocystis jiroveci Pneumonia secondary to tyrosine kinase inhibitor with blinatumomab therapy: A case report.

One of the main challenges in compiling the complete collection of protein antigens from pathogens for the selection of vaccine candidates or intervention targets is to acquire a broad enough representation of them to be recognized by the highly diversified immunoglobulin repertoire in human populations. Dried serum spot sampling (DSS) retains a large repertoire of circulating immunoglobulins from each individual that can be representative of a population, according to the sample size. In this work, shotgun proteomics of an infectious pathogen based on DSS sampling coupled with IgM immunoprecipitation, liquid chromatography-mass spectrometry (LC-MS/MS), and bioinformatic analyses was combined to characterize the circulating IgM antigenome. Serum samples from a malaria endemic region at different clinical statuses were studied to optimize IgM binding efficiency and antibody leaching by varying serum/immunomagnetic bead ratios and elution conditions. The method was validated using Plasmodium falciparum extracts identifying 110 of its IgM-reactive antigens while minimizing the presence of human proteins and antibodies. Furthermore, the IgM antigen recognition profile differentiated between malaria-infected and noninfected individuals at the time of sampling. We conclude that a shotgun proteomics approach offers advantages in providing a high-throughput, reliable, and clean way to identify IgM-recognized antigens from trace amounts of serum. The mass spectrometry raw data and metadata have been deposited with ProteomeXchange via MassIVE with the PXD identifier PXD043800.

Shotgun Characterization of the Circulating IgM Antigenome of an Infectious Pathogen by Immunocapture-LC-MS/MS from Dried Serum Spots.

Strengths and limitations of molecular diagnostics for Pneumocystis jirovecii pneumonia

Pneumocystis jirovecii is the most common cause of fungal pneumonia in children under the age of 2 years. However, the inability to culture and propagate this organism has hampered the acquisition of a fungal genome as well as the development of recombinant antigens to conduct seroprevalence studies. In this study, we performed proteomics on Pneumocystis-infected mice and used the recent P. murina and P. jirovecii genomes to prioritize antigens for recombinant protein expression. We focused on a fungal glucanase due to its conservation among fungal species. We found evidence of maternal IgG to this antigen, followed by a nadir in pediatric samples between 1 and 3 months of age, followed by an increase in prevalence over time consistent with the known epidemiology of Pneumocystis exposure. Moreover, there was a strong concordance of anti-glucanase responses and IgG against another Pneumocystis antigen, PNEG_01454. Taken together, these antigens may be useful tools for Pneumocystis seroprevalence and seroconversion studies.

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Novel Pneumocystis Antigens for Seroprevalence Studies

Pneumocystis is the most common fungal pulmonary infection in children under the age of 5 years. In children with primary immunodeficiency, Pneumocystis often presents at 3–6 months of age, a time period that coincides with the nadir of maternal IgG and when IgM is the dominant Ig isotype. Because B cells are the dominant antigen-presenting cells for Pneumocystis, we hypothesized the presence of fungal-specific IgMs in humans and mice and that these IgM specificities would predict T cell antigens. We detected fungal-specific IgMs in human and mouse sera and utilized immunoprecipitation to determine whether any antigens were similar across donors. We then assessed T cell responses to these antigens and found anti-Pneumocystis IgM in WT mice, Aicda–/– mice, and in human cord blood. Immunoprecipitation of Pneumocystis murina with human cord blood identified shared antigens among these donors. Using class II MHC binding prediction, we designed peptides with these antigens and identified robust peptide-specific lung T cell responses after P. murina infection. After mice were immunized with 2 of the antigens, adoptive transfer of vaccine-elicited CD4+ T cells showed effector activity, suggesting that these antigens contain protective Pneumocystis epitopes. These data support the notion that germline-encoded IgM B cell receptors are critical in antigen presentation and T cell priming in early Pneumocystis infection.

/pdf/germline-igm-predicts-t-cell-immunity-to-pneumocystis-16d3sgff.pdf

Germline IgM predicts T cell immunity to Pneumocystis

ABSTRACT Pneumocystis pneumonia (PCP) is a serious fungal lung infection caused by Pneumocystis jirovecii in immunosuppressed individuals. The lack of viable in vitro/ex vivo PCP models has greatly hindered the progress in studying the biology of these fungi, the host/pathogen interactions, as well as antifungal susceptibility testing. In this study, we show the utility of precision-cut lung slices (PCLS) to support the survival of Pneumocystis murina in vitro. We cultured PCLS tissue derived from wild type and immunocompromised mice with a P. murina inoculum in submerged or air-liquid interface models for up to 14 days. We isolated total RNA from the cultured lung tissues at days 0, 3, 7, 10, and 14 and analyzed for the expression of host lung genes and P. murina genes (Gsc1 for asci and Sp for trophs) by real-time quantitative PCR. When cultured in media alone, P. murina died gradually within a few days. However, when cultured on PCLS, both the troph and ascus forms survived throughout the incubation period of 2 weeks. Moreover, immunohistochemistry staining of P. murina inoculated PCLS sections using polyclonal anti-Pneumocystis sera and showed evidence of fungal aggregation and possible biofilm formation. Additionally, in vitro (PCLS) antibiotic susceptibility testing using commonly used antifungal drugs confirmed successful targeting of the troph and ascus forms by trimethoprim sulfamethoxazole and the ascus form by echinocandins. IMPORTANCE Our study reveals the potential of precision-cut lung slices as an ex vivo platform to study the growth/survival of Pneumocystis spp. that can facilitate the development of new anti-fungal drugs. Our study reveals the potential of precision-cut lung slices as an ex vivo platform to study the growth/survival of Pneumocystis spp. that can facilitate the development of new anti-fungal drugs.

Precision-cut lung slices as an ex vivo model to study Pneumocystis murina survival and antimicrobial susceptibility

CRISPR-mediated detection of Pneumocystis transcripts in bronchoalveolar, oropharyngeal, and serum specimens for Pneumocystis pneumonia diagnosis

Pneumocystis jirovecii is an important etiological agent of pneumonia that is underdiagnosed due to the inability to culture the organism. The 2019 PERCH study identified Pneumocystis as the top fungal cause of pneumonia in HIV-negative children using a PCR cutoff of 104 copies of Pneumocystis per mL of sample in nasopharyngeal/oropharyngeal (NP/OP) specimens. Given that Pneumocystis consists of an environmental ascus form and a trophic from (the latter is the form that attaches to the lung epithelium), it is possible that life-form-specific molecular assays may be useful for diagnosis. However, to accomplish this goal, these assays require genotypic information, as the current fungal genomic data are largely from the US and Europe. To genotype Pneumocystis across the globe, we developed an NGS-based genotyping assay focused on genes expressed in asci as well as trophs using PERCH throat swabs from Africa, Bangladesh, and Thailand, as well as North American samples. The NGS panel reliably detected 21 fungal targets in these samples and revealed unique genotypes in genes expressed in trophs, including Meu10, an ascospore assembly gene; two in mitochondrial gene ATP8, and the intergenic region between COX1 and ATP8. This assay can be used for enhanced Pneumocystis epidemiology to study outbreaks but also permits more accurate RT-CPR- or CRISPR-based assays to be performed to improve the non-bronchoscopic diagnosis of this under-reported fungal pathogen.

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Targeted NGS-Based Analysis of Pneumocystis jirovecii Reveals Novel Genotypes

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