Multidrug-Resistant Bacterial Colonization and Infections in Large Retrospective Cohort of Mechanically Ventilated COVID-19 Patients1.

Few data are available on incidence of multidrug-resistant organism (MDRO) colonization and infections in mechanically ventilated patients, particularly during the COVID-19 pandemic. We retrospectively evaluated all patients admitted to the COVID-19 intensive care unit (ICU) of Hub Hospital in Milan, Italy, during October 2020‒May 2021. Microbiologic surveillance was standardized with active screening at admission and weekly during ICU stay. Of 435 patients, 88 (20.2%) had MDROs isolated ≤48 h after admission. Of the remaining patients, MDRO colonization was diagnosed in 173 (51.2%), MDRO infections in 95 (28.1%), and non-MDRO infections in 212 (62.7%). Non-MDRO infections occurred earlier than MDRO infections (6 days vs. 10 days; p<0.001). Previous exposure to antimicrobial drugs within the ICU was higher in MDRO patients than in non-MDRO patients (116/197 [58.9%] vs. 18/140 [12.9%]; p<0.001). Our findings might serve as warnings for future respiratory viral pandemics and call for increased measures of antimicrobial stewardship and infection control.

Distal and proximal hypoxia response elements cooperate to regulate organ-specific erythropoietin gene expression.

While it is well-established that distal hypoxia response elements (HREs) regulate hypoxia-inducible factor (HIF) target genes such as erythropoietin (Epo), an interplay between multiple distal and proximal (promoter) HREs has not been described so far. Hepatic Epo expression is regulated by a HRE located downstream of the EPO gene, but this 3' HRE is dispensable for renal EPO gene expression. We previously identified a 5' HRE and could show that both HREs direct exogenous reporter gene expression. Here, we show that whereas in hepatic cells the 3' but not the 5' HRE is required, in neuronal cells both the 5' and 3' HREs contribute to endogenous Epo induction. Moreover, two novel putative HREs were identified in the EPO promoter. In hepatoma cells HIF interacted mainly with the distal 3' HRE, but in neuronal cells HIF most strongly bound the promoter, to a lesser extent the 3' HRE, and not at all the 5' HRE. Interestingly, mutation of either of the two distal HREs abrogated HIF binding to the 3' and promoter HREs. These results suggest that a canonical functional HRE can recruit multiple, not necessarily HIF, transcription factors to mediate HIF binding to different distant HREs in an organ-specific manner.

Successful recovery from severe COVID-19 pneumonia after kidney transplantation: The interplay between immunosuppression and novel therapy including tocilizumab.

Although immunosuppressed patients may be more prone to SARS-CoV-2 infection with atypical presentation, long-term immunosuppression therapy may provide some sort of protection for severe clinical complications of COVID-19. The interaction between immunosuppression and new antiviral drugs in the treatment of transplanted patients contracting COVID-19 has not yet been fully investigated. Moreover, data regarding the optimal management of these patients are still very limited. We report a case of the successful recovery from severe COVID-19 of a kidney-transplanted patient treated with hydroxychloroquine, lopinavir/ritonavir, steroid, and tocilizumab.

Generation of renal Epo-producing cell lines by conditional gene tagging reveals rapid HIF-2 driven Epo kinetics, cell autonomous feedback regulation, and a telocyte phenotype.

Erythropoietin (Epo) is essential for erythropoiesis and is mainly produced by the fetal liver and the adult kidney following hypoxic stimulation. Epo regulation is commonly studied in hepatoma cell lines, but differences in Epo regulation between kidney and liver limit the understanding of Epo dysregulation in polycythaemia and anaemia. To overcome this limitation, we have generated a novel transgenic mouse model expressing Cre recombinase specifically in the active fraction of renal Epo-producing (REP) cells. Crossing with reporter mice confirmed the inducible and highly specific tagging of REP cells, located in the corticomedullary border region where there is a steep drop in oxygen bioavailability. A novel method was developed to selectively grow primary REP cells in culture and to generate immortalized clonal cell lines, called fibroblastoid atypical interstitial kidney (FAIK) cells. FAIK cells show very early hypoxia-inducible factor (HIF)-2α induction, which precedes Epo transcription. Epo induction in FAIK cells reverses rapidly despite ongoing hypoxia, suggesting a cell autonomous feedback mechanism. In contrast, HIF stabilizing drugs resulted in chronic Epo induction in FAIK cells. RNA sequencing of three FAIK cell lines derived from independent kidneys revealed a high degree of overlap and suggests that REP cells represent a unique cell type with properties of pericytes, fibroblasts, and neurons, known as telocytes. These novel cell lines may be helpful to investigate myofibroblast differentiation in chronic kidney disease and to elucidate the molecular mechanisms of HIF stabilizing drugs currently in phase III studies to treat anemia in end-stage kidney disease.

Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping.

A crucial step in the cellular adaptation to oxygen deficiency is the binding of hypoxia-inducible factors (HIFs) to hypoxia response elements (HREs) of oxygen-regulated genes. Genome-wide HIF-1α/2α/ß DNA-binding studies revealed that the majority of HREs reside distant to the promoter regions, but the function of these distal HREs has only been marginally studied in the genomic context. We used chromatin immunoprecipitation (ChIP), gene editing (TALEN) and chromosome conformation capture (3C) to localize and functionally characterize a 82 kb upstream HRE that solely drives oxygen-regulated expression of the newly identified HIF target gene PAG1. PAG1, a transmembrane adaptor protein involved in Src signalling, was hypoxically induced in various cell lines and mouse tissues. ChIP and reporter gene assays demonstrated that the -82 kb HRE regulates PAG1, but not an equally distant gene further upstream, by direct interaction with HIF. Ablation of the consensus HRE motif abolished the hypoxic induction of PAG1 but not general oxygen signalling. 3C assays revealed that the -82 kb HRE physically associates with the PAG1 promoter region, independent of HIF-DNA interaction. These results demonstrate a constitutive interaction between the -82 kb HRE and the PAG1 promoter, suggesting a physiologically important rapid response to hypoxia.

The galactocerebrosidase enzyme contributes to maintain a functional neurogenic niche during early post-natal CNS development.

We report a novel role for the lysosomal galactosylceramidase (GALC), which is defective in globoid cell leukodystrophy (GLD), in maintaining a functional post-natal subventricular zone (SVZ) neurogenic niche. We show that proliferation/self-renewal of neural stem cells (NSCs) and survival of their neuronal and oligodendroglial progeny are impaired in GALC-deficient mice. Using drugs to modulate inflammation and gene transfer to rescue GALC expression and activity, we show that lipid accumulation resulting from GALC deficiency acts as a cell-autonomous pathogenic stimulus in enzyme-deficient NSCs and progeny before upregulation of inflammatory markers, which later sustain a non-cell-autonomous dysfunction. Importantly, we provide evidence that supply of functional GALC provided by neonatal intracerebral transplantation of NSCs ameliorates the functional impairment in endogenous SVZ cells. Insights into the mechanism/s underlying GALC-mediated regulation of early post-natal neurogenic niches improve our understanding of the multi-component pathology of GLD. The occurrence of a restricted period of SVZ neurogenesis in infancy supports the implications of our study for the development of therapeutic strategies to treat this severe pediatric neurodegenerative disorder.

Urinary secretion and extracellular aggregation of mutant uromodulin isoforms.

Uromodulin is exclusively expressed in the thick ascending limb and is the most abundant protein secreted in urine where it is found in high-molecular-weight polymers. Its biological functions are still elusive, but it is thought to play a protective role against urinary tract infection, calcium oxalate crystal formation, and regulation of water and salt balance in the thick ascending limb. Mutations in uromodulin are responsible for autosomal-dominant kidney diseases characterized by defective urine concentrating ability, hyperuricemia, gout, tubulointerstitial fibrosis, renal cysts, and chronic kidney disease. Previous in vitro studies found retention in the endoplasmic reticulum as a common feature of all uromodulin mutant isoforms. Both in vitro and in vivo we found that mutant isoforms partially escaped retention in the endoplasmic reticulum and reached the plasma membrane where they formed large extracellular aggregates that have a dominant-negative effect on coexpressed wild-type protein. Notably, mutant uromodulin excretion was detected in patients carrying uromodulin mutations. Thus, our results suggest that mutant uromodulin exerts a gain-of-function effect that can be exerted by both intra- and extracellular forms of the protein.

Androglobin, a chimeric mammalian globin, is required for male fertility.

Spermatogenesis is a highly specialized differentiation process driven by a dynamic gene expression program and ending with the production of mature spermatozoa. Whereas hundreds of genes are known to be essential for male germline proliferation and differentiation, the contribution of several genes remains uncharacterized. The predominant expression of the latest globin family member, androglobin (Adgb), in mammalian testis tissue prompted us to assess its physiological function in spermatogenesis. Adgb knockout mice display male infertility, reduced testis weight, impaired maturation of elongating spermatids, abnormal sperm shape, and ultrastructural defects in microtubule and mitochondrial organization. Epididymal sperm from Adgb knockout animals display multiple flagellar malformations including coiled, bifid or shortened flagella, and erratic acrosomal development. Following immunoprecipitation and mass spectrometry, we could identify septin 10 (Sept10) as interactor of Adgb. The Sept10-Adgb interaction was confirmed both in vivo using testis lysates and in vitro by reciprocal co-immunoprecipitation experiments. Furthermore, the absence of Adgb leads to mislocalization of Sept10 in sperm, indicating defective manchette and sperm annulus formation. Finally, in vitro data suggest that Adgb contributes to Sept10 proteolysis in a calmodulin-dependent manner. Collectively, our results provide evidence that Adgb is essential for murine spermatogenesis and further suggest that Adgb is required for sperm head shaping via the manchette and proper flagellum formation.

Urinary uromodulin carries an intact ZP domain generated by a conserved C-terminal proteolytic cleavage.

Uromodulin (or Tamm-Horsfall protein) is the most abundant protein in human urine under physiological conditions. Little is known about the molecular mechanism of uromodulin secretion. By extensive Mass Spectrometry analyses we mapped the C-termini of human and murine urinary proteins demonstrating that urinary uromodulin is generated by a conserved C-terminal proteolytic cleavage and retains its entire ZP domain.

Estrogen-dependent downregulation of hypoxia-inducible factor (HIF)-2α in invasive breast cancer cells.

The involvement of estrogen (E2) and hypoxia in tumor progression is well established. Hypoxia has been reported to activate and degrade estrogen receptor alpha (ERα) in breast cancer cells. Furthermore, E2 has been shown to regulate hypoxia-inducible factor (HIF)-1α protein, but its role in HIF-2α regulation remains largely unexplored. In this study, we found that both HIF-2α mRNA and protein were down-regulated in ER positive but not ER negative breast cancer cells upon treatment with E2. The analysis of 690 samples derived from 608 mixed and 82 triple-negative breast cancer patients revealed that high nuclear HIF-2α tumor levels are associated with a worse prognosis specifically in human epidermal growth factor receptor 2 (HER2) and hormone receptor positive patients. Consistently, ERα/HER2 positive breast cancer cells displayed less pronounced downregulation of HIF-2α by E2. Experiments using a histone deacetylase inhibitor indicate that the E2 mediated decrease in HIF-2α mRNA is due to transcriptional repression. A functional estrogen response element (ERE) was identified in the first intron of the gene encoding HIF-2α (EPAS1), suggesting transcriptional co-repressor recruitment by ERα. Our results demonstrate a novel modulation of HIF-2α in breast cancer cells, explaining the opposing regulation between HIF-1α and HIF-2α in hormone-responsive breast cancer.

Validation of a novel index of hemorrhage using a lower body negative pressure shock model.

BACKGROUND: Vital signs are late indicators of blood loss in trauma patients. Indexed Heart to Arm Time (iHAT) is a non-invasive index based on a modified pulse transit time (mPTT) indexed to the time between R waves on the electrocardiogram (RR interval). We aimed to investigate how early iHAT is able to detect central hypovolemia during the progression from mild to severe simulated hemorrhage induced by applying lower body negative pressure (LBNP). METHODS: Thirty healthy volunteers were enrolled. Central hypovolemia was induced by application of increasing LBNP from 0 to -80 mmHg. At every step, non-invasive blood pressure, heart rate, cardiac echo Doppler measurements and iHAT were recorded. RESULTS: Aortic flow Velocity Time Integral (VTI) reduction from 21.8±3.7 (baseline) to 11.2±3 cm (-70 mmHg) (P<0.001) was progressive with LBNP increase and represented a significant change in stroke volume and preload and induced an increase in heart rate from 69±2 to 107±4 bpm. iHAT increased from 34.2±4.65% (baseline) to 53.9±14.34% (-80 mmHg), P<0.001. The increase in iHAT became significant after -30 mmHg level was reached, corresponding to 500-1000 mL blood loss. CONCLUSIONS: iHAT measures both the reduction in preload and the parabolic heart rate increase due to the linear decrease in stroke volume. iHAT was able to detect a progressive central volume loss in a model of hemorrhage in healthy volunteers undergoing LBNP. A rising trend in iHAT can be a useful marker for progressive volume loss during moderate to severe bleeding.

Induction of long noncoding RNA MALAT1 in hypoxic mice.

Long thought to be "junk DNA", in recent years it has become clear that a substantial fraction of intergenic genomic DNA is actually transcribed, forming long noncoding RNA (lncRNA). Like mRNA, lncRNA can also be spliced, capped, and polyadenylated, affecting a multitude of biological processes. While the molecular mechanisms underlying the function of lncRNAs have just begun to be elucidated, the conditional regulation of lncRNAs remains largely unexplored. In genome-wide studies our group and others recently found hypoxic transcriptional induction of a subset of lncRNAs, whereof nuclear-enriched abundant/autosomal transcript 1 (NEAT1) and metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) appear to be the lncRNAs most ubiquitously and most strongly induced by hypoxia in cultured cells. Hypoxia-inducible factor (HIF)-2 rather than HIF-1 seems to be the preferred transcriptional activator of these lncRNAs. For the first time, we also found strong induction primarily of MALAT1 in organs of mice exposed to inspiratory hypoxia. Most abundant hypoxic levels of MALAT1 lncRNA were found in kidney and testis. In situ hybridization revealed that the hypoxic induction in the kidney was confined to proximal rather than distal tubular epithelial cells. Direct oxygen-dependent regulation of MALAT1 lncRNA was confirmed using isolated primary kidney epithelial cells. In summary, high expression levels and acute, profound hypoxic induction of MALAT1 suggest a hitherto unrecognized role of this lncRNA in renal proximal tubular function.

The serine protease hepsin mediates urinary secretion and polymerisation of Zona Pellucida domain protein uromodulin.

Uromodulin is the most abundant protein in the urine. It is exclusively produced by renal epithelial cells and it plays key roles in kidney function and disease. Uromodulin mainly exerts its function as an extracellular matrix whose assembly depends on a conserved, specific proteolytic cleavage leading to conformational activation of a Zona Pellucida (ZP) polymerisation domain. Through a comprehensive approach, including extensive characterisation of uromodulin processing in cellular models and in specific knock-out mice, we demonstrate that the membrane-bound serine protease hepsin is the enzyme responsible for the physiological cleavage of uromodulin. Our findings define a key aspect of uromodulin biology and identify the first in vivo substrate of hepsin. The identification of hepsin as the first protease involved in the release of a ZP domain protein is likely relevant for other members of this protein family, including several extracellular proteins, as egg coat proteins and inner ear tectorins.

Cytokine production in women with antiretroviral treatment-associated breast fat accumulation and limb wasting.

OBJECTIVE: To test the cytokine production of peripheral blood mononuclear cells in a group of HIV-infected women with breast enlargement and lower limb wasting while receiving antiretroviral therapy (ART) including a protease inhibitor. DESIGN: Case-control study including 20 women with fat tissue alterations and 20 matched controls treated with comparable ART. METHODS: Adipose tissue alterations (ATA) were defined by increased breast size (> or = 2 bra sizes) accompanied by lower limb fat wasting. A randomly selected subset of patients underwent analyses including: dual energy X-ray absorptiometry, metabolic and endocrine assays, in vitro cytokine production testing [interferon-gamma, interleukin (IL)-2, IL-4, IL-10, IL-12, tumor necrosis factor-alpha (TNF-alpha)] after appropriate stimulation; T-cell phenotyping, T-helper function after stimulation with either tetanus toxoid, influenza antigen, allogeneic peripheral blood lymphocytes, and phytohemagglutinin. Endocrinological study included the determination of plasma concentrations of prolactin, growth hormone, testosterone, adrenocorticotropic hormone, cortisol and C-peptide. RESULTS: In vitro production of IL-12 was higher (P = 0.0001), and TNF-alpha (P = 0.0093) and IL-10 (P < 0.0001) production were lower in stimulated peripheral blood mononuclear cells of ATA-positive women compared with ATA-negative women. ATA-positive women also showed a better response to tetanus toxoid (P = 0.021) and a lower median fluorescence intensity of CD14/DR (P=0.033). Plasma C-peptide values were higher in ATA-positive women compared with ATA-negative women (P = 0.033), even if in the normal range (< 4 ng/ml) in all but one of the ATA-positive patients. CONCLUSION: HIV-1-infected women who developed breast enlargement and lower limb fat wasting while receiving ART had a favorable immunological profile with efficient IL-12 production and T-helper function, and with TNF-a production in the range of a HIV-negative reference population. These findings suggest that the rescue of some immune functions under ART may be involved in the pathogenesis of this particular adipose tissue disorder.

Analysis of uromodulin polymerization provides new insights into the mechanisms regulating ZP domain-mediated protein assembly.

Uromodulin is the most abundant protein secreted in urine, in which it is found as a high-molecular-weight polymer. Polymerization occurs via its zona pellucida (ZP) domain, a conserved module shared by many extracellular eukaryotic proteins that are able to assemble into matrices. In this work, we identified two motifs in uromodulin, mapping in the linker region of the ZP domain and in between protein cleavage and glycosylphosphatidylinositol (GPI)-anchoring sites, which regulate its polymerization. Indeed, mutations in either module led to premature intracellular polymerization of a soluble uromodulin isoform, demonstrating the inhibitory role of these motifs for ZP domain-mediated protein assembly. Proteolytic cleavage separating the external motif from the mature monomer is necessary to release the inhibitory function and allow protein polymerization. Moreover, we report absent or abnormal assembly into filaments of GPI-anchored uromodulin mutated in either the internal or the external motif. This effect is due to altered processing on the plasma membrane, demonstrating that the presence of the two modules has not only an inhibitory function but also can positively regulate protein polymerization. Our data expand previous knowledge on the control of ZP domain function and suggest a common mechanism regulating polymerization of ZP domain proteins.

Defective intracellular trafficking of uromodulin mutant isoforms.

Medullary cystic kidney disease/familial juvenile hyperuricemic nephropathy (MCKD/FJHN) are autosomal dominant renal disorders characterized by tubulo-interstitial fibrosis, hyperuricemia and medullary cysts. They are caused by mutations in the gene encoding uromodulin, the most abundant protein in urine. Uromodulin (or Tamm-Horsfall protein) is a glycoprotein that is exclusively expressed by epithelial tubular cells of the thick ascending limb of Henle's loop and distal convoluted tubule. To date, 37 different uromodulin mutations have been described in patients with MCKD/FJHN. Interestingly, 60% of them involve one of the 48 conserved cysteine residues. We have previously shown that cysteine-affecting mutations could lead to partial endoplasmic reticulum (ER) retention. In this study, as a further step in understanding uromodulin biology in health and disease, we provide the first extensive study of intracellular trafficking and subcellular localization of wild-type and mutant uromodulin isoforms. We analyzed a set of 12 different uromodulin mutations that were representative of the different kind of mutations identified so far by different experimental approaches (immunofluorescence, electron microscopy, biochemistry and in vivo imaging) in transiently transfected HEK293 and Madin-Darby canine kidney cells. We assessed protein processing in the secretory pathway and could demonstrate that although to different extent, all uromodulin mutations lead to defective ER to Golgi protein transport, suggesting a common pathogenetic mechanism in MCKD/FJHN.