Pseudoxanthoma elasticum (PXE) patients and heterozygous carriers have decreased plasma PPi levels which correlate with the (cardio)vascular phenotype.

Introduction Pseudoxanthoma elasticum (PXE) is an autosomal recessive soft tissue calcification disorder caused by biallelic mutations in ABCC6. In patients decreased levels of inorganic pyrophosphate (PPi) have been linked to the formation of ectopic mineralizations. However, no data has been reported on PPi levels in heterozygous carriers. Moreover, it is unknown whether a correlation exists between PPi levels and the ABCC6 genotype or the phenotype of patients. Methods A total of 127 citrated blood samples (91 from 62 PXE patients, 22 from 21 carriers and 14 from 14 controls) was collected and processed. Plasma PPi levels were measured via ATPase luminescence assays and samples were internally calibrated. The ABCC6 gene was screened for variants. The diagnosis of PXE was confirmed by the presence of 2 (likely) pathogenic variants (scored as C3 to C5 according to Verschuere et al. 2020) and/or histopathological changes. PXE phenotypes were assessed using the Phenodex scoring (PS) system at the time the first blood sample was obtained for a patient. Two sets of data were analyzed using SPSS26. First an analysis was performed where all available samples were considered [Bulk]. Concomitantly, a smaller subset (i.e. the samples obtained at Phenodex scoring [Single]) was analyzed. Differences in age, sex and PPi levels between the 3 cohorts were analyzed via ANOVA and T-Test. Correlations between PPi, PS, age and sex were analyzed via Spearman’s correlation analysis. After samples were subdivided based on 3 mutation archetypes: erroneous mRNA transcript (D: deletion, frameshift, splicing), nonsense (N) and missense (M) variants, genotype-PPi correlations were assessed via multiple ANOVA analyses based on more stringent variant selection (C3 - C5, C3 [Subset Likely Pathogenic] - C5, C4 - C5, C5). Results Compared to controls, patients and carriers had respectively ±49% and ±22% less PPi (P<0.001), though overlap in the sample ranges exists between the groups. Interestingly, PPi levels were lower in males compared to females in PXE patients (Bulk: P=0.011; Single: P=0.071), but not in the other cohorts. No significant differences in age between cohorts were observed; however, in the patient cohort PPi levels increased significantly with age (P=0.0015, r=0.279). Moreover, a significant inverse correlation between PPi levels and cardiac PS scores was identified (P=0.041, r=-0.263) as well as a weak inverse association between PPi and vascular PS (P=0.09). No correlation was found with skin or eye symptoms. A significant difference in PPi levels between D+M and N+M clusters was found during bulk, but not single, analysis (P<0.01). Conclusion PPi plasma levels are significantly decreased in patients and carriers and may thus also contribute to the subclinical or partial phenotype seen in the latter. Importantly, there is a region of overlap between both and with controls, suggesting PPi not to be the only relevant pro-mineralization factor in at least a subset of PXE patients. In PXE patients PPi levels appear to be influenced by sex and - to some extent unexpectedly - significantly increase with age. Whether there is a solid correlation with the ABCC6 genotype remains to be confirmed. Though unrelated to skin and eye disease, the PPi levels are inversely correlated with cardio(vascular) burden in PXE patients, making PPi promising as a cardiovascular biomarker in PXE. Reference Verschuere et al. 2020: PMID 3287393

Chlamydia trachomatis L2c infection in a porcine model produced urogenital pathology and failed to induce protective immune responses against re-infection

The current study was designed to evaluate the pathogenesis, pathology and immune response of female genital tract infection with Chlamydia trachomatis L2c, the most recently discovered lymphogranuloma venereum strain, using a porcine model of sexually transmitted infections. Pigs were mock infected, infected once or infected and re-infected intravaginally, and samples were obtained for chlamydial culture, gross and microscopic pathology, and humoral and cell-mediated immunity. Intravaginal inoculation of pigs with this bacterium resulted in an infection that was confined to the urogenital tract, where inflammation and pathology were caused that resembled what is seen in human infection. Re-infection resulted in more severe gross pathology than primary infection, and chlamydial colonization of the urogenital tract was similar for primary infected and re- infected pigs. This indicates that primary infection failed to induce protective immune responses against re-infection. Indeed, the proliferative responses of mononuclear cells from blood and lymphoid tissues to C. trachomatis strain L2c were never statistically different among groups, suggesting that C. trachomatis-specific lymphocytes were not generated following infection or re-infection. Nevertheless, anti-chlamydial antibodies were elicited in sera and vaginal secretions after primary infection and re-infection, clearly resulting in a secondary systemic and mucosal antibody response. While primary infection did not protect against reinfection, the porcine model is relevant for evaluating immune and pathogenic responses for emerging and known C. trachomatis strains to advance drug and/or vaccine development in humans

Cellular and molecular biomarkers indicate premature aging in pseudoxanthoma elasticum patients

The molecular processes of aging are very heterogenic and not fully understood. Studies on rare progeria syndromes, which display an accelerated progression of physiological aging, can help to get a better understanding. Pseudoxanthoma elasticum (PXE) caused by mutations in the ATP-binding cassette sub-family C member 6 (ABCC6) gene shares some molecular characteristics with such premature aging diseases. Thus, this is the first study trying to broaden the knowledge of aging processes in PXE patients. In this study, we investigated aging associated biomarkers in primary human dermal fibroblasts and sera from PXE patients compared to healthy controls. Determination of serum concentrations of the aging biomarkers eotaxin-1 (CCL11), growth differentiation factor 11 (GDF11) and insulin-like growth factor 1 (IGF1) showed no significant differences between PXE patients and healthy controls. Insulin-like growth factor binding protein 3 (IGFBP3) showed a significant increase in serum concentrations of PXE patients older than 45 years compared to the appropriate control group. Tissue specific gene expression of GDF11 and IGFBP3 were significantly decreased in fibroblasts from PXE patients compared to normal human dermal fibroblasts (NHDF). IGFBP3 protein concentration in supernatants of fibroblasts from PXE patients were decreased compared to NHDF but did not reach statistical significance due to potential gender specific variations. The minor changes in concentration of circulating aging biomarkers in sera of PXE patients and the significant aberrant tissue specific expression seen for selected factors in PXE fibroblasts, suggests a link between ABCC6 deficiency and accelerated aging processes in affected peripheral tissues of PXE patients

Genetic deletion of Abcc6 disturbs cholesterol homeostasis in mice

Genetic studies link adenosine triphosphate-binding cassette transporter C6 (ABCC6) mutations to pseudoxanthoma elasticum (PXE). ABCC6 sequence variations are correlated with altered HDL cholesterol levels and an elevated risk of coronary artery diseases. However, the role of ABCC6 in cholesterol homeostasis is not widely known. Here, we report reduced serum cholesterol and phytosterol levels in Abcc6-deficient mice, indicating an impaired sterol absorption. Ratios of cholesterol precursors to cholesterol were increased, confirmed by upregulation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase (Hmgcr) expression, suggesting activation of cholesterol biosynthesis in Abcc6-/- mice. We found that cholesterol depletion was accompanied by a substantial decrease in HDL cholesterol mediated by lowered ApoA-I and ApoA-II protein levels and not by inhibited lecithin-cholesterol transferase activity. Additionally, higher proprotein convertase subtilisin/kexin type 9 (Pcsk9) serum levels in Abcc6-/- mice and PXE patients and elevated ApoB level in knockout mice were observed, suggesting a potentially altered very low-density lipoprotein synthesis. Our results underline the role of Abcc6 in cholesterol homeostasis and indicate impaired cholesterol metabolism as an important pathomechanism involved in PXE manifestation

Assessing immunogenicity barriers of the HIV-1 envelope trimer

Understanding the balance between epitope shielding and accessibility on HIV-1 envelope (Env) trimers is essential to guide immunogen selection for broadly neutralizing antibody (bnAb) based vaccines. To investigate the antigenic space of Env immunogens, we created a strategy based on synthetic, high diversity, Designed Ankyrin Repeat Protein (DARPin) libraries. We show that DARPin Antigenicity Analysis (DANA), a purely in vitro screening tool, has the capability to extrapolate relevant information of antigenic properties of Env immunogens. DANA screens of stabilized, soluble Env trimers revealed that stronger trimer stabilization led to the selection of highly mutated DARPins with length variations and framework mutations mirroring observations made for bnAbs. By mimicking heterotypic prime-boost immunization regimens, DANA may be used to select immunogen combinations that favor the selection of trimer-reactive binders. This positions DANA as a versatile strategy for distilling fundamental antigenic features of immunogens, complementary to preclinical immunogenicity testing

Nonlinear optical microscopy is a novel tool for the analysis of cutaneous alterations in pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE, OMIM 264800) is a rare autosomal recessive disorder with ectopic mineralization and fragmentation of elastin fibers. It is caused by mutations of the ABCC6 gene that leads to decreased serum levels of inorganic pyrophosphate (PPi) anti-mineralization factor. The occurrence of severe complications among PXE patients highlights the importance of early diagnosis so that prompt multidisciplinary care can be provided to patients. We aimed to examine dermal connective tissue with nonlinear optical (NLO) techniques, as collagen emits second-harmonic generation (SHG) signal, while elastin can be excited by two-photon excitation fluorescence (TPF). We performed molecular genetic analysis, ophthalmological and cardiovascular assessment, plasma PPi measurement, conventional histopathological examination, and ex vivo SHG and TPF imaging in five patients with PXE and five age- and gender-matched healthy controls. Pathological mutations including one new variant were found in the ABCC6 gene in all PXE patients and their plasma PPi level was significantly lower compared with controls. Degradation and mineralization of elastin fibers and extensive calcium deposition in the mid-dermis was visualized and quantified together with the alterations of the collagen structure in PXE. Our data suggests that NLO provides high-resolution imaging of the specific histopathological features of PXE-affected skin. In vivo NLO may be a promising tool in the assessment of PXE, promoting early diagnosis and follow-up

Macrophages retain hematopoietic stem cells in the spleen via VCAM-1

Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)[superscript +] macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE[superscript −/−] mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques

Long-term low-level ambient air pollution exposure and risk of lung cancer - A pooled analysis of 7 European cohorts.

BACKGROUND/AIM: Ambient air pollution has been associated with lung cancer, but the shape of the exposure-response function - especially at low exposure levels - is not well described. The aim of this study was to address the relationship between long-term low-level air pollution exposure and lung cancer incidence. METHODS: The "Effects of Low-level Air Pollution: a Study in Europe" (ELAPSE) collaboration pools seven cohorts from across Europe. We developed hybrid models combining air pollution monitoring, land use data, satellite observations, and dispersion model estimates for nitrogen dioxide (NO2), fine particulate matter (PM2.5), black carbon (BC), and ozone (O3) to assign exposure to cohort participants' residential addresses in 100 m by 100 m grids. We applied stratified Cox proportional hazards models, adjusting for potential confounders (age, sex, calendar year, marital status, smoking, body mass index, employment status, and neighborhood-level socio-economic status). We fitted linear models, linear models in subsets, Shape-Constrained Health Impact Functions (SCHIF), and natural cubic spline models to assess the shape of the association between air pollution and lung cancer at concentrations below existing standards and guidelines. RESULTS: The analyses included 307,550 cohort participants. During a mean follow-up of 18.1 years, 3956 incident lung cancer cases occurred. Median (Q1, Q3) annual (2010) exposure levels of NO2, PM2.5, BC and O3 (warm season) were 24.2 µg/m3 (19.5, 29.7), 15.4 µg/m3 (12.8, 17.3), 1.6 10-5m-1 (1.3, 1.8), and 86.6 µg/m3 (78.5, 92.9), respectively. We observed a higher risk for lung cancer with higher exposure to PM2.5 (HR: 1.13, 95% CI: 1.05, 1.23 per 5 µg/m3). This association was robust to adjustment for other pollutants. The SCHIF, spline and subset analyses suggested a linear or supra-linear association with no evidence of a threshold. In subset analyses, risk estimates were clearly elevated for the subset of subjects with exposure below the EU limit value of 25 µg/m3. We did not observe associations between NO2, BC or O3 and lung cancer incidence. CONCLUSIONS: Long-term ambient PM2.5 exposure is associated with lung cancer incidence even at concentrations below current EU limit values and possibly WHO Air Quality Guidelines