Application of crispr/cas9-based reverse genetics in leishmania braziliensis: Conserved roles for hsp100 and hsp23

The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR–Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene (eGFP) and two L. braziliensis single-copy genes (HSP23 and HSP100). We obtained homozygous Cas9-free HSP23-and HSP100-null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null mutants. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. major HSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of L. braziliensis by CRISPR–Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite’s biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis.Alexander von Humboldt-StiftungRevisión por pare

Differential risks of psoriatic arthritis development in patients with varied psoriasis manifestations: a sex- and ethnicity-specific analysis

ObjectivesThis study investigated psoriatic arthritis (PsA) risk across varied psoriasis manifestations, considering sex and ethnicity.MethodsUsing TriNetX, a federated database encompassing over 120 million electronic health records (EHRs), we performed global retrospective cohort studies. Psoriasis vulgaris (Pso), pustulosis palmoplantaris (PPP), and generalized pustular psoriasis (GPP) cohorts were retrieved using ICD-10 codes. Propensity score matching, incorporating age, sex, and ethnicity, was employed. An alternative propensity matching model additionally included established PsA risk factors.ResultsWe retrieved data from 486 (Black or African American-stratified, GPP) to 35,281 (Pso) EHRs from the US Collaborative Network. Significant PsA risk variations emerged: Pso carried the highest risk [hazard ratio (HR) 87.7, confidence interval (CI) 63.4–121.1, p < 0.001], followed by GPP (HR 26.8, CI 6.5–110.1, p < 0.0001), and PPP (HR 15.3, CI 7.9–29.5, p < 0.0001). Moreover, we identified significant sex- and ethnicity-specific disparities in PsA development. For instance, compared to male Pso patients, female Pso patients had an elevated PsA risk (HR 1.1, CI 1.1–1.2, p = 0.002). Furthermore, White Pso patients had a higher likelihood of developing PsA compared to their Black or African American counterparts (HR 1.3, CI 1.04–1.7, p = 0.0244). We validated key findings using alternative propensity matching strategies and independent databases.ConclusionThis study delineates nuanced PsA risk profiles across psoriasis forms, highlighting the pivotal roles of sex and ethnicity. Integrating these factors into PsA risk assessments enables tailored monitoring and interventions, potentially impacting psoriasis patient care quality

Cutaneous lupus erythematosus is associated with an increased risk of cardiac and vascular diseases: a large-scale, propensity-matched global retrospective cohort studyResearch in context

Summary: Background: Autoimmune skin diseases can expedite various systemic sequelae involving other organs. Although limited to the skin, cutaneous lupus erythematosus (CLE) was noted to be associated with thromboembolic diseases. However, small cohort sizes, partially discrepant outcomes, missing data on CLE subtypes, and incomplete risk assessment limits these findings. Methods: The Global Collaborative Network of TriNetX provides access to medical records of more than 120 million patients worldwide. We used TriNetX to elucidate the risk for cardiac and vascular diseases after diagnosis of CLE, and its subtypes chronic discoid (DLE) and subacute cutaneous lupus erythematosus (SCLE). We included 30,315 CLE, 27,427 DLE, and 1613 SCLE patients. We performed propensity-matched cohort studies determining the risk to develop cardiac and vascular diseases (ICD10CM:I00-99) following diagnosis of CLE, DLE, or SCLE. Patients with systemic lupus erythematosus were excluded. Findings: We document that CLE and its subtype DLE but less so SCLE are associated with a higher risk for various cardiac and vascular diseases. This included predominantly thromboembolic events such as pulmonary embolism, cerebral infarction, and acute myocardial infarction, but also peripheral vascular disease and pericarditis. For example, the hazard ratio of arterial embolism and thrombosis was 1.399 (confidence interval: 1.230–1.591, p < 0.0001) following CLE diagnosis. The study is limited by retrospective data collection and reliance on ICD10-disease classification. Interpretation: CLE and its major subtype DLE are associated with an increased risk for the development of a wide range of cardiac and vascular diseases. Funding: This research was funded by Deutsche Forschungsgemeinschaft (EXC 2167, CSSL/CS01-2022) and the Excellence-Chair Program of the State of Schleswig-Holstein

Application of CRISPR/Cas9-based reverse genetics in Leishmania braziliensis : conserved roles for HSP100 and HSP23

The protozoan parasite Leishmania (Viannia) braziliensis (L. braziliensis) is the main cause of human tegumentary leishmaniasis in the New World, a disease affecting the skin and/or mucosal tissues. Despite its importance, the study of the unique biology of L. braziliensis through reverse genetics analyses has so far lagged behind in comparison with Old World Leishmania spp. In this study, we successfully applied a cloning-free, PCR-based CRISPR&ndash;Cas9 technology in L. braziliensis that was previously developed for Old World Leishmania major and New World L. mexicana species. As proof of principle, we demonstrate the targeted replacement of a transgene (eGFP) and two L. braziliensis single-copy genes (HSP23 and HSP100). We obtained homozygous Cas9-free HSP23- and HSP100-null mutants in L. braziliensis that matched the phenotypes reported previously for the respective L. donovani null mutants. The function of HSP23 is indeed conserved throughout the Trypanosomatida as L. majorHSP23 null mutants could be complemented phenotypically with transgenes from a range of trypanosomatids. In summary, the feasibility of genetic manipulation of L. braziliensis by CRISPR&ndash;Cas9-mediated gene editing sets the stage for testing the role of specific genes in that parasite&rsquo;s biology, including functional studies of virulence factors in relevant animal models to reveal novel therapeutic targets to combat American tegumentary leishmaniasis

Psoriasis Management Challenges Regarding Difficult-to-Treat Areas: Therapeutic Decision and Effectiveness

Psoriasis is not optimally controlled in spite of newly developed treatments, possibly due to the difficulty of objectively quantifying the disease&rsquo;s severity, considering the limitations of the clinical scores used in clinical practice. A major challenge addresses difficult-to-treat areas, especially in the absence of significant body surface involvement. It is controversial whether the severity evaluation of patients with several affected areas (having at least one difficult-to-treat area) should be done differently from current methods. Scores used for special areas (PSSI, NAPSI and ESIF) allow an accurate assessment of disease severity in difficult-to-treat areas, but the issue of whether to integrate these scores into PASI, BSA or DLQI remains. The review&rsquo;s purpose resides in providing an overview of the main current issues in determining psoriasis severity in patients with psoriasis in difficult-to-treat areas and suggesting possible solutions for the optimal integration of the area assessment in current scores: severity can be either established according to the highest calculated score (PASI or PSSI or NAPSI or ESIF) or by adding a correction factor in the calculation of PASI for special areas

Tryptophan degradation as a systems phenomenon in inflammation – an analysis across 13 chronic inflammatory diseasesResearch in context

Summary: Background: Chronic inflammatory diseases (CIDs) are systems disorders that affect diverse organs including the intestine, joints and skin. The essential amino acid tryptophan (Trp) can be broken down to various bioactive derivatives important for immune regulation. Increased Trp catabolism has been observed in some CIDs, so we aimed to characterise the specificity and extent of Trp degradation as a systems phenomenon across CIDs. Methods: We used high performance liquid chromatography and targeted mass spectrometry to assess the serum and stool levels of Trp and Trp derivatives. Our retrospective study incorporates both cross-sectional and longitudinal components, as we have included a healthy population as a reference and there are also multiple observations per patient over time. Findings: We found reduced serum Trp levels across the majority of CIDs, and a prevailing negative relationship between Trp and systemic inflammatory marker C-reactive protein (CRP). Notably, serum Trp was low in several CIDs even in the absence of measurable systemic inflammation. Increases in the kynurenine-to-Trp ratio (Kyn:Trp) suggest that these changes result from increased degradation along the kynurenine pathway. Interpretation: Increases in Kyn:Trp indicate the kynurenine pathway as a major route for CID-related Trp metabolism disruption and the specificity of the network changes indicates excessive Trp degradation relative to other proteogenic amino acids. Our results suggest that increased Trp catabolism is a common metabolic occurrence in CIDs that may directly affect systemic immunity. Funding: This work was supported by the DFG Cluster of Excellence 2167 “Precision medicine in chronic inflammation” (KA, SSchr, PR, BH, SWa), the BMBF (e:Med Juniorverbund “Try-IBD” 01ZX1915A and 01ZX2215, the e:Med Network iTREAT 01ZX2202A, and GUIDE-IBD 031L0188A), EKFS (2020_EKCS.11, KA), DFG RU5042 (PR, KA), and Innovative Medicines Initiative 2 Joint Undertakings (“Taxonomy, Treatments, Targets and Remission”, 831434, “ImmUniverse”, 853995, “BIOMAP”, 821511)

Detection of intracellular parasites within the ring mask.

<p>Macrophages were cultured in 96-well cultures in the presence (plus <i>L</i>. <i>major</i>, right row) or absence of <i>L</i>. <i>major</i> parasites (w/o <i>L</i>. <i>major</i>, left row). After 72h a DAPI staining was performed to visualize nuclei of the mammalian cells and the DNA rich areas of the parasites simultaneously. Single channel greyscale pictures were processed by TissueQuest software. The ring masks were created as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139866#pone.0139866.g001" target="_blank">Fig 1</a> and material and methods. (A) The ring mask (highlighted in blue) visualizes the cytoplasmatic area of a representative macrophage that was cultured in the absence of parasites. (B) One representative macrophage infected with <i>L</i>. <i>major</i> parasites is shown including the ring mask which is highlighted in blue. Visually determined DAPI positive signals are marked with yellow arrows. For the detection of parasites within the ring masks a virtual channel of the parasite-associated fluorochrome (in our case DAPI) has to be created (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139866#pone.0139866.s002" target="_blank">S2C Fig</a>). The following instrument settings were used: Use ring mask: yes, interior radius: -0,31 μm, exterior radius: 12,74 μm, use identification mask: no, use nucleus mask: no, background threshold: 5. (C) One algorithm developed for the detection of weak signals recognizes false positive signals (yellow squares) within the ring mask (the cell shown in A is highlighted in blue) of macrophages that are not infected. (D) False positive signals (yellow squares) are created. The representative (highlighted in blue) macrophage harbors more than 30 parasites, whereas no more than 10 can be visually determined (see B). (E) The algorithm that was developed to detect weak signals recognizes no false positive signals in the ring mask of macrophages that are not infected. (F) All parasites within infected macrophages are recognized (see yellow squares and yellow arrows in (B)). Representative pictures out of 3 experiments are shown. Bar = 10 μm.</p

TissueQuest-based characterization of infected macrophages.

<p>Macrophages were cultured in 96-well plates in the presence of <i>L</i>. <i>major</i> parasites (parasite/macrophage ratio 5:1). (A) The infected macrophages were determined automatically by the TissueQuest software. The circle diagrams depict the distribution of infected (black) and not infected macrophages (grey). The infection rate is indicated within the circle diagram. A representative experiment visualizing the infection rate after measuring 310 or 11237 macrophages is shown. (B) The box-plots depict the TissueQuest based quantification of the average number of intracellular parasites per macrophage after analysis of 237 or 8639 macrophages. The gray boxes indicate the range between the first and third quartile. The horizontal lines indicate the median. The whiskers visualize the spread of data. The red bracket highlights the additional information that was generated after measuring 8639 infected macrophages compared to analyzing 237 infected macrophages (***<0.0001). The data shown in (A) and (B) are representative for three experiments. (C) Macrophages were analyzed regarding their number of intracellular parasites and the amount of macrophages (y-axis) harboring the indicated number of parasites (x-axis) are shown. Data are presented in box-blots. The circle diagram highlights the number of macrophages harboring 1–6 (light gray), 7–12 (dark gray) and 12–20 (black) parasites. Data are representative for three experiments. (D) Validation of TissueQuest-based quantification of parasites by real-time PCR was performed. Macrophages were infected with different numbers of parasites per host cell (1:1 and 5:1) and 96 hours post infection the average number of parasites per macrophage (right y-axis) and the number of <i>L</i>. <i>major</i> parasites per β-actin was determined (left y-axis) (*<0,01, *** 0,0001; n = 3; mean +/- SD).</p

Nuclei detection and ring mask calculation by TissueQuest.

<p>Single channel greyscale pictures were processed by TissueQuest software. (A) The nuclei detection was performed according the following parameters (nuclei size: 10, remove small objects: 1, remove weakly stained objects: 1, automated background: no, automated threshold: 5, virtual channel: no, post processing order: remove, merge and Remove labels i) smaller then: 30 μm, larger then: 100 μm, weaker then: 137, stronger then: do not use; use merging rules: no). The detected nuclei are automatically surrounded by a red line. (B) The ring masks were created according the following parameters (interior radius: -0,31 μm, exterior radius; 12,74 μm, use identification cell mask: no, use nucleus mask: no, background threshold: 5; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139866#pone.0139866.s001" target="_blank">S1 Fig</a>). The white insert highlights one infected macrophage. The white arrows depict some of the parasites within the ring mask. (C) The area within the ring mask is highlighted in yellow and represents the region of interest in which the screening of parasites can be performed automatically. Representative pictures out of 3 experiments are shown. Bar = 10μm.</p

Phenotypical analysis of macrophages harboring different numbers of parasites.

<p>Macrophages were cultured in 96-well plates in the presence of <i>L</i>. <i>major</i> parasites (parasite/macrophage ratio 5:1). After 72 hours the cells were stained with F4/80^Cy5, CD11b^biotinylated + Streptavidin^AF546. Macrophage nuclei and parasite DNA rich areas were stained with DAPI. (A) The TissueQuest-based analysis of cells and intracellular parasites are presented. A histogram plot depicts the number of parasites within the macrophages (x-axis) and the number of macrophages harboring the indicated (see x-axis) number of <i>Leishmania</i> parasites. Cells within the highlighted gates were further analyzed regarding the expression of CD11b and F4/80. (B) The mean intensities of CD11b or F4/80 are plotted representing macrophages harboring no (#0), one (#1), six (#6) or ten (#10) parasites. Statistical analyses were performed with GraphPad Prism using the nonparametic Mann-Whitney test (p**<0,01, p*<0,05; red horizontal line represents the median). Every single dot represents one individual analyzed nucleated cell. One representative set of data out of two experiments is shown.</p