Search

Background: The contribution of gene-environment interactions that lead to excessive aggression is poorly understood. Environmental stressors and mutations of the gene encoding tryptophan hydroxylase-2 (TPH2) are known to influence aggression. For example, TPH2 null mutant mice (Tph2−/−) are naturally highly aggressive, while heterozygous mice (Tph2+/−) lack a behavioral phenotype and are considered endophenotypically normal. Here we sought to discover whether an environmental stressor would affect the phenotype of the genetically ‘susceptible’ heterozygous mice (Tph2+/−). Methods: Tph2+/− male mice or Tph2+/+ controls were subjected to a five-day long rat exposure stress paradigm. Brain serotonin metabolism and the expression of selected genes encoding serotonin receptors, AMPA receptors, and stress markers were studied. Results: Stressed Tph2+/− mice displayed increased levels of aggression and social dominance, whereas Tph2+/+ animals became less aggressive and less dominant. Brain tissue concentrations of serotonin, its precursor hydroxytryptophan and its metabolite 5-hydroxyindoleacetic acid were significantly altered in all groups in the prefrontal cortex, striatum, amygdala, hippocampus and dorsal raphe after stress. Compared to non-stressed animals, the concentration of 5-hydroxytryptophan was elevated in the amygdala though decreased in the other brain structures. The overexpression of the AMPA receptor subunit, GluA2, and downregulation of 5-HT6 receptor, as well as overexpression of c-fos and glycogen-synthase-kinase-3β (GSK3-β), were found in most structures of the stressed Tph2+/− mice. Limitations: Rescue experiments would help to verify causal relationships of reported changes. Conclusions: The interaction of a partial TPH2 gene deficit with stress results in pathological aggression and molecular changes, and suggests that the presence of genetic susceptibility can augment aggression in seemingly resistant phenotypes. © 2020 The Authors

Outbred female rats were exposed to inhalation of lead oxide nanoparticle aerosol produced right then and there at a concentration of 1.30 ± 0.10 mg/m3 during 5 days for 4 h a day in a nose-only setup. A control group of rats were sham-exposed in parallel under similar conditions. Even this short-time exposure of a relatively low level was associated with nanoparticles retention demonstrable by transmission electron microscopy in the lungs and the olfactory brain. Some impairments were found in the organism’s status in the exposed group, some of which might be considered lead-specific toxicological outcomes (in particular, increase in reticulocytes proportion, in δ-aminolevulinic acid (δ-ALA) urine excretion, and the arterial hypertension’s development). © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

The development of novel and effective methods for the isolation of chitin, which remains one of the fundamental aminopolysaccharides within skeletal structures of diverse marine invertebrates, is still relevant. In contrast to numerous studies on chitin extraction from crustaceans, mollusks and sponges, there are only a few reports concerning its isolation from corals, and especially black corals (Antipatharia). In this work, we report the stepwise isolation and identification of chitin from Cirrhipathes sp. (Antipatharia, Antipathidae) for the first time. The proposed method, aiming at the extraction of the chitinous scaffold from the skeleton of black coral species, combined a well-known chemical treatment with in situ electrolysis, using a concentrated Na2SO4 aqueous solution as the electrolyte. This novel method allows the isolation of α-chitin in the form of a microporous membrane-like material. Moreover, the extracted chitinous scaffold, with a well-preserved, unique pore distribution, has been extracted in an astoundingly short time (12 h) compared to the earlier reported attempts at chitin isolation from Antipatharia corals. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline and neuropathological features, including abnormal deposition of amyloid-β (Aβ) peptides, intracellular neurofibrillary tangles, and neuronal death. Identifying therapeutics which can reduce memory deficits at an early stage of the disease has the advantage of slowing or even reversing disease progression before irreversible brain damage has occurred. Consequently, in this study, we investigated the ability of the histone deacetylase inhibitor sodium butyrate (NaB) to attenuate memory deficits in the 5xFAD mouse model of AD following a 12-week feeding regimen. 5xFAD mice demonstrate a unique time course of Aβ pathology, developing Aβ plaques as early as 2 months. Male mice were assigned to either a control diet or a NaB-supplemented diet which was administered at either 5 mg/kg/day, or 15 mg/kg/day for 12 weeks (each group, N = 15). Supplementation commenced at an early disease stage (8-10 weeks of age). Behavioral testing (contextual and cued fear conditioning) was undertaken, and brain Aβ levels measured, at the end of the 12-week intervention. NaB had profound effects on Aβ levels and on associative learning and cognitive functioning. A 40% reduction in brain Aβ levels and a 25% increase in fear response in both the cued and contextual testing was observed in the NaB-treated animals compared to the control group. These findings suggest that NaB warrants further investigation as a potential therapeutic agent in the treatment of cognitive deficits associated with early stages of AD. © 2020 - IOS Press and the authors. All rights reserved.

The field of spatial transcriptomics is rapidly expanding, and with it the repertoire of available technologies. However, several of the transcriptome-wide spatial assays do not operate on a single cell level, but rather produce data comprised of contributions from a – potentially heterogeneous – mixture of cells. Still, these techniques are attractive to use when examining complex tissue specimens with diverse cell populations, where complete expression profiles are required to properly capture their richness. Motivated by an interest to put gene expression into context and delineate the spatial arrangement of cell types within a tissue, we here present a model-based probabilistic method that uses single cell data to deconvolve the cell mixtures in spatial data. To illustrate the capacity of our method, we use data from different experimental platforms and spatially map cell types from the mouse brain and developmental heart, which arrange as expected., QC 20201230

Genetic mutations in FUS, a DNA/RNA-binding protein, are associated with inherited forms of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). A novel transgenic FUS[1-359]-tg mouse line recapitulates core hallmarks of human ALS in the spinal cord, including neuroinflammation and neurodegeneration, ensuing muscle atrophy and paralysis, as well as brain pathomorphological signs of FTLD. However, a question whether FUS[1-359]-tg mouse displays behavioural and brain pro-inflammatory changes characteristic for the FTLD syndrome was not addressed. Here, we studied emotional, social and cognitive behaviours, brain markers of inflammation and plasticity of pre-symptomatic FUS[1-359]-tg male mice, a potential FTLD model. These animals displayed aberrant behaviours and altered brain expression of inflammatory markers and related pathways that are reminiscent to the FTLD-like syndrome. FTLD-related behavioural and molecular Journal of Cellular and Molecular Medicine features were studied in the pre-symptomatic FUS[1-359]-tg mice that received standard or new ALS treatments, which have been reported to counteract the ALS-like syndrome in the mutants. We used anti-ALS drug riluzole (8 mg/kg/d), or anti-inflammatory drug, a selective blocker of cyclooxygenase-2 (celecoxib, 30 mg/kg/d) for 3 weeks, or a single intracerebroventricular (i.c.v.) infusion of human stem cells (Neuro-Cells, 500 000-CD34+), which showed anti-inflammatory properties. Signs of elevated anxiety, depressive-like behaviour, cognitive deficits and abnormal social behaviour were less marked in FUS-tg–treated animals. Applied treatments have normalized protein expression of interleukin-1β (IL-1β) in the prefrontal cortex and the hippocampus, and of Iba-1 and GSK-3β in the hippocampus. Thus, the pre-symptomatic FUS[1-359]-tg mice demonstrate FTLD-like abnormalities that are attenuated by standard and new ALS treatments, including Neuro-Cell preparation. © 2020 The Authors. Journal

Ascending bacterial urinary tract infections can cause epididymo-orchitis. In the cauda epididymidis, this frequently leads to persistent tissue damage. Less coherent data is available concerning the functional consequences of epididymo-orchitis on testis and caput epididymidis. This in vivo study addresses the functional and spatial differences in responsiveness of murine epididymis and testis to infection with uropathogenic Escherichia coli (UPEC). Whole transcriptome analysis (WTA) was performed on testis, caput, corpus and cauda epididymidis of adult C57BL/6 J wildtype mice. Following UPEC-induced epididymo-orchitis in these mice, epididymal and testicular tissue damage was evaluated histologically and semi-quantitatively at 10 days and 31 days post-inoculation. Expression of inflammatory markers and candidate antimicrobial genes were analysed by RT-qPCR. WTA revealed distinct differences in gene signatures between caput and cauda epididymidis, particularly amonst immunity-related genes. Cellular and molecular signs of testicular inflammation and disruption of spermatogenesis were noticed at day 10, but recovery was observed by day 31. In contrast to the cauda, the caput epididymidis did not reveal any signs of gross morphological damage or presence of pro-inflammatory processes despite confirmed infection. In contrast to beta-defensins, known UPEC-associated antimicrobial peptides (AMP), like Lcn2, Camp and Lypd8, were inherently highly expressed or upregulated in the caput following infection, potentially allowing an early luminal protection from UPEC. At the time points investigated, the caput epididymidis was protected from any obvious infection/inflammation-derived tissue damage. Studies addressing earlier time-points will conclude whether in the caput epididymidis a pro-inflammatory response is indeed not essential for effective protection from UPEC.Copyright © 2020 The Author(s) 2020. Published by Oxford University Press on behalf of the European Society

Objective Lung hypoplasia associated with congenital diaphragmatic hernia (CDH) results in respiratory insufficiency and pulmonary hypertension after birth. We have investigated whether aerating the lung before removing placental support (physiologically based cord clamping (PBCC)), improves the cardiopulmonary transition in lambs with a CDH. Methods At =138 days of gestational age, 17 lambs with surgically induced left-sided diaphragmatic hernia (=d80) were delivered via caesarean section. The umbilical cord was clamped either immediately prior to ventilation onset (immediate cord clamping (ICC); n=6) or after achieving a target tidal volume of 4 mL/kg, with a maximum delay of 10 min (PBCC; n=11). Lambs were ventilated for 120 min and physiological changes recorded. Results Pulmonary blood flow (PBF) increased following ventilation onset in both groups, but was 19-fold greater in PBCC compared with ICC lambs at cord clamping (19+/-6.3 vs 1.0+/-0.5 mL/min/kg, p<0.001). Cerebral tissue oxygenation was higher in PBCC than ICC lambs during the first 10 min after cord clamping (59%+/-4% vs 30%+/-5%, p<0.001). PBF was threefold higher (23+/-4 vs 8+/-2 mL/min/kg, p=0.01) and pulmonary vascular resistance (PVR) was threefold lower (0.6+/-0.1 vs 2.2+/-0.6 mm Hg/(mL/min), p<0.001) in PBCC lambs compared with ICC lambs at 120 min after ventilation onset. Conclusions Compared with ICC, PBCC prevented the severe asphyxia immediately after birth and resulted in a higher PBF due to a lower PVR, which persisted for at least 120 min after birth in CDH lambs.Copyright © Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.

Background: There are fewer preclinical models of prostate cancer compared to other common tumours. New models that represent the diverse features of castrate-sensitive and castration-resistant prostate cancer (CRPC) are required for thorough preclinical testing of novel treatments. Therefore, the goal of the Melbourne Urological Research Alliance (MURAL) is to develop patient-derived xenografts (PDXs) spanning the clinical trajectory of prostate cancer. Method(s): We grafted >200 surgery, biopsy or rapid autopsy samples into testosterone-supplemented or castrated male NSG mice. Actively growing tumours were serially transplanted or grown as explants or organoids. PDXs were analysed using RNAseq, targeted genomic sequencing and histopathology review. Result(s): We previously reported 4 serially transplantable PDXs (Lawrence, et al., 2018, European Urology). Now we have established ~30 additional models spanning treatment naive primary disease to CRPC. PDXs of CRPC were often from soft tissue metastases of patients who had failed docetaxel, cabazitaxel, enzalutamide, abiraterone and other contemporary treatments. Accordingly, they had diverse mechanisms of resistance, including AR mutations, genomic structural rearrangements, gene amplifications and expression of AR variants. In addition, several PDXs had AR-null phenotypes, including small cell prostate cancer. All PDXs closely reflected the genomic, transcriptomic and histopathological characteristics of the original patient tumours. As renewable sources of tissue, the PDXs could also be grown as ex vivo slice cultures and in vitro organoids, providing complementary models with different timescales and endpoints. Conclusion(s): We have developed a new collection of castrate-sensitive and castrate-resistant PDXs of prostate cancer, providing diverse tumours for preclinical testing of candidate treatments.

MCL-1 is one of the most frequently amplified genes in cancer, facilitating tumor initiation and maintenance and enabling resistance to anti-tumorigenic agents including the BCL-2 selective inhibitor venetoclax. The expression of MCL-1 is maintained via P-TEFb-mediated transcription, where the kinase CDK9 is a critical component. Consequently, we developed a series of potent small-molecule inhibitors of CDK9, exemplified by the orally active A-1592668, with CDK selectivity profiles that are distinct from related molecules that have been extensively studied clinically. Short-term treatment with A-1592668 rapidly downregulates RNA pol-II (Ser 2) phosphorylation resulting in the loss of MCL-1 protein and apoptosis in MCL-1-dependent hematologic tumor cell lines. This cell death could be attenuated by either inhibiting caspases or overexpressing BCL-2 protein. Synergistic cell killing was also observed between A-1592668 or the related analog A-1467729, and venetoclax in a number of hematologic cell lines and primary NHL patient samples. Importantly, the CDK9 inhibitor plus venetoclax combination was well tolerated in vivo and demonstrated efficacy superior to either agent alone in mouse models of lymphoma and AML. These data indicate that CDK9 inhibitors could be highly efficacious in tumors that depend on MCL-1 for survival or when used in combination with venetoclax in malignancies dependent on MCL-1 and BCL-2.Copyright © 2019, The Author(s).

Gastric cancer is associated with chronic inflammation (gastritis) triggered by persistent Helicobacter pylori (H. pylori) infection. Elevated tyrosine phosphorylation of the latent transcription factor STAT3 is a feature of gastric cancer, including H. pylori-infected tissues, and aligns with nuclear transcriptional activity. However, the transcriptional role of STAT3 serine phosphorylation, which promotes STAT3-driven mitochondrial activities, is unclear. Here, by coupling serine-phosphorylated (pS)-STAT3-deficient Stat3SA/SA mice with chronic H. felis infection, which mimics human H. pylori infection in mice, we reveal a key role for pS-STAT3 in promoting Helicobacter-induced gastric pathology. Immunohistochemical staining for infiltrating immune cells and expression analyses of inflammatory genes revealed that gastritis was markedly suppressed in infected Stat3SA/SA mice compared with wild-type mice. Stomach weight and gastric mucosal thickness were also reduced in infected Stat3SA/SA mice, which was associated with reduced proliferative potential of infected Stat3SA/SA gastric mucosa. The suppressed H. felis-induced gastric phenotype of Stat3SA/SA mice was phenocopied upon genetic ablation of signaling by the cytokine IL-11, which promotes gastric tumorigenesis via STAT3. pS-STAT3 dependency by Helicobacter coincided with transcriptional activity on STAT3-regulated genes, rather than mitochondrial and metabolic genes. In the gastric mucosa of mice and patients with gastritis, pS-STAT3 was constitutively expressed irrespective of Helicobacter infection. Collectively, these findings suggest an obligate requirement for IL-11 signaling via constitutive pS-STAT3 in Helicobacter-induced gastric carcinogenesis.Copyright © 2020 American Society for Investigative Pathology

Background & Aims: The protease plasmin is an important wound healing factor, but it is not clear how it affects gastrointestinal infection-mediated damage, such as that resulting from Clostridioides difficile. We investigated the role of plasmin in C difficile-associated disease. This bacterium produces a spore form that is required for infection, so we also investigated the effects of plasmin on spores. Method(s): C57BL/6J mice expressing the precursor to plasmin, the zymogen human plasminogen (hPLG), or infused with hPLG were infected with C difficile, and disease progression was monitored. Gut tissues were collected, and cytokine production and tissue damage were analyzed by using proteomic and cytokine arrays. Antibodies that inhibit either hPLG activation or plasmin activity were developed and structurally characterized, and their effects were tested in mice. Spores were isolated from infected patients or mice and visualized using super-resolution microscopy; the functional consequences of hPLG binding to spores were determined. Result(s): hPLG localized to the toxin-damaged gut, resulting in immune dysregulation with an increased abundance of cytokines (such as interleukin [IL] 1A, IL1B, IL3, IL10, IL12B, MCP1, MP1A, MP1B, GCSF, GMCSF, KC, TIMP-1), tissue degradation, and reduced survival. Administration of antibodies that inhibit plasminogen activation reduced disease severity in mice. C difficile spores bound specifically to hPLG and active plasmin degraded their surface, facilitating rapid germination. Conclusion(s): We found that hPLG is recruited to the damaged gut, exacerbating C difficile disease in mice. hPLG binds to C difficile spores, and, upon activation to plasmin, remodels the spore surface, facilitating rapid spore germination. Inhibitors of plasminogen activation might be developed for treatment of C difficile or other infection-mediated gastrointestinal diseases.Copyright © 2020

Macrophage-mediated renal injury promotes the development of diabetic nephropathy. Blockade of chemokine (C-C motif) receptor 2 (CCR2) inhibits kidney macrophage accumulation and early glomerular damage in diabetic animals. This study tested early and late interventions with a CCR2 antagonist (CCR2A) in a model of progressive diabetic glomerulosclerosis and determined whether CCR2A provides added benefit over conventional treatment with an angiotensin-converting enzyme inhibitor (ACEi). Diabetes was induced in hypertensive endothelial nitric oxide synthase (Nos3)-deficient mice by administration of five low-dose streptozotocin (STZ) injections daily. Groups of diabetic Nos3- /- mice received a CCR2A (30 mg.kg- 1.day- 1 PF-04634817 in chow) as an early intervention (weeks 2-15 after STZ). The late intervention (weeks 8-15 after STZ) involved PF-04634817 alone, ACEi (captopril in water 10 mg.kg- 1. day- 1) alone, or combined ACEi + CCR2A. Control diabetic and nondiabetic Nos3- /- mice received normal chow and water. Early intervention with a CCR2A inhibited kidney inflammation and glomerulosclerosis, albuminuria, podocyte loss, and renal function impairment but not hypertension in diabetic Nos3- /- mice. Late intervention with a CCR2A also inhibited kidney inflammation, glomerulosclerosis, and renal dysfunction but did not affect albuminuria. ACEi alone suppressed hypertension and albuminuria and partially reduced podocyte loss and glomerulosclerosis but did not affect renal dysfunction. Compared with ACEi alone, the combined late intervention with ACEi + CCR2A provided better protection against kidney damage (inflammation, glomerulosclerosis, and renal function impairment) but not albuminuria. In conclusion, this study demonstrates that combining CCR2A and ACEi provides broader and superior renal protection than ACEi alone in a model of established diabetic glomerulosclerosis with hypertension.Copyright © 2019 the American Physiological Society

Background: Accumulating evidence suggests that dysregulation of neutrophil extracellular traps (NETs) could be associated with the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA) vasculitis. Peptidyl arginine deiminases (PADs) are enzymes that generate citrullinated proteins, which are pro-inflammatory in both innate and adaptive immune responses. Citrullination of histone 3 (H3Cit) by PAD2 and PAD4 facilitates generation of both macrophage extracellular traps (METs) and NETs. BB-Clamidine is a second generation pan (PAD) inhibitor which reduces citrullination of H3Cit and subsequent NET formation. This study investigates the contribution of NETs in the pathogenesis of experimental anti-myeloperoxidase ANCA associated glomerulonephritis (MPO-ANCA GN) and the therapeutic possibility of BB-Cl amidine disrupting NET formation in vivo, and attenuating anti-MPO GN. Method(s): Experimental anti-MPO GN was induced in C57BL/6 mice by myeloperoxidase (MPO) immunisation and GN triggered using a subnephritogenic dose of anti-glomerular basement membrane globulin. BB-Cl-amidine treatment (1mg/kg/daily, n=6 or vehicle, n=7) was administered to disrupt NET formation after the establishment of MPO autoimmunity until termination of the experiment. Result(s): NET accumulation identified by co-localisation of extracellular DNA, H3Cit, PAD4, and MPO, was prominent in glomeruli of untreated (vehicle) mice compared to BB-Cl-amidine treated mice (2.1 +/- 0.4 vs 0.4 +/- 0.1, P=0.004). Histological assessment of kidneys demonstrated prominent glomerular segmental necrosis in the untreated group versus a reduction in mice receiving BB-Cl-amidine (88 +/- 3 vs 41 +/- 3, P=0.004). BBCl-amidine significantly attenuated glomerular leukocyte infiltration; CD4 T cells (1.2 +/- 0.3 vs 0.3 +/- 0.1, P=0.03), macrophages (1 +/- 0.1 vs 0.4 +/- 0.06, P=0.008) and neutrophils (3 +/- 0.5 vs 1.6 +/-0.3, P=0.03), compared to untreated mice. BB-Cl-amidine treated mice had increased numbers of CD

Background: Anti-cytokine monoclonal antibody (mAb) therapies have been effective in many autoimmune diseases but have not been successfully trialled in MPO-ANCA associated vasculitis. This study assessed the efficacy of blocking key CD4+ Th1 subset signature effector cytokines, TNFalpha and IFNgamma in MPO-ANCA GN. Assessing therapeutic efficacy of these anti-cytokine mAbs is now complicated by our recent discovery that Th subset dominance during the development of MPO autoimmunity is biphasic with initial transient Th17 dominance followed by persistent Th1 responses. Method(s): Anti-MPO autoimmunity was induced in C57BL/6 mice by MPO immunization and GN triggered using anti-GBM Ig during early and late development of anti-MPO autoimmunity, and GN assessed 4 days later (days 20 and 32, respectively). mAb treatment began 4hrs post GN triggering. Result(s): Administration of anti-TNFalpha mAb early in anti-MPO development (day 20) had no effect on kidney injury compared with vehicle treated controls: albuminuria [5.7+/-1.7 vs 5.5+/-1.7 mg/24hr, P=0.9]; glomerular segmental necrosis [GSN: 50+/-4 vs 43+/-3, P=0.1]. Similarly, anti-IFNgamma mAb was ineffective in attenuating GN at this timepoint [GSN: 48+/-6 vs 45+/-3, P=0.53]. Failure of these treatments is concordant with our observation that early developing anti-MPO autoimmunity is Th17 dominant. In contrast, anti-TNFalpha therapy during established anti-MPO GN (day 32) markedly attenuated kidney injury [GSN: 27+/-2 vs 50+/-4, P=0.01]. TNFalpha blockade acts locally in the kidney as systemic MPO specific IFNgamma and IL-17 recall responses from lymph nodes draining MPO immunization sites were similar to vehicle treated controls. Neutralizing IFNgamma at this late timepoint induced a phenotypic switch from Th1 responses to a protective Th2 with increased in serum MPO-ANCA [1.19+/-0.25 vs 0.48+/-0.13 OD450nm, P=0.02], increased IL-4 production from MPO challenged LN cells and increased the proportion of activated M2 m

Background & Aims: Helicobacter pylori induces strong inflammatory responses that are directed at clearing the infection, but if not controlled, these responses can be harmful to the host. We investigated the immune-regulatory effects of the innate immune molecule, nucleotide-binding oligomerization domain-like receptors (NLR) family CARD domain-containing 5 (NLRC5), in patients and mice with Helicobacter infection. Method(s): We obtained gastric biopsies from 30 patients in Australia. We performed studies with mice that lack NLRC5 in the myeloid linage (Nlrc5moKO) and mice without Nlrc5 gene disruption (controls). Some mice were gavaged with H pylori SS1 or Helicobacter felis; 3 months later, stomachs, spleens, and sera were collected, along with macrophages derived from bone marrow. Human and mouse gastric tissues and mouse macrophages were analyzed by histology, immunohistochemistry, immunoblots, and quantitative polymerase chain reaction. THP-1 cells (human macrophages, controls) and NLRC5-/- THP-1 cells (generated by CRISPR-Cas9 gene editing) were incubated with Helicobacter and gene expression and production of cytokines were analyzed. Result(s): Levels of NLRC5 messenger RNA were significantly increased in gastric tissues from patients with H pylori infection, compared with patients without infection (P <.01), and correlated with gastritis severity (P <.05). H pylori bacteria induced significantly higher levels of chemokine and cytokine production by NLRC5-/- THP-1 macrophages than by control THP-1 cells (P <.05). After 3 months of infection with H felis, Nlrc5mo-KO mice developed gastric hyperplasia (P <.0001), splenomegaly (P <.0001), and increased serum antibody titers (P <.01), whereas control mice did not. Nlrc5mo-KO mice with chronic H felis infection had increased numbers of gastric B-cell follicles expressing CD19 (P <.0001); these follicles had features of mucosa-associated lymphoid tissue lymphoma. We identified B-cell-activating factor as a protein

Background: Tubular activation of the c-Jun amino-terminal kinase (JNK) pathway is prominent in most forms of acute and progressive tubulointerstitial damage, including renal ischaemia/reperfusion (I/R) injury. Both Jnk1 and Jnk2 genes are expressed in most cells of the kidney resulting in considerable redundancy. Combined blockade of JNK1/2 is known to be protective in renal I/R injury; however, the relative contribution of each isoform is unknown. The aim of this study is to determine the relative contribution of JNK1 versus JNK2 in renal I/R injury. Method(s): Preferential pharmacological inhibition of JNK1 (CC90001) was compared to mice with global deletion of Jnk1 or Jnk2 and mice with combined global Jnk2 deletion plus Jnk1 deletion in proximal tubular cells (Jnk-PT mice). Bilateral warm renal ischaemia injury was induced with vascular clamps and animals killed 24hr after reperfusion. Controls were sham operated. Sprague-Dawley rats (n=8-10/group) were treated 3 times with CC-90001 (10mg/kg) or vehicle starting 1 hour prior to surgery. Renal I/R injury studies were also performed in Jnk1-/- (n=10), Jnk2-/-(n=8), Jnk PT (n=8) and wild type (WT) mice (n=10). Result(s): Treatment with CC-90001 provided significant protection in rat I/R injury (8+/-0.5 vs 20+/-2 fold increase in serum creatinine (sCR), drug versus vehicle, respectively; P<0.001). In a separate study, Jnk1-/- mice also showed significant protection from I/R injury compared to WT mice (4+/-0.5 vs 8+/-1 fold increase in sCr respectively; p=0.01); however, this was not replicated in Jnk2-/-mice (16+/-0.5 vs 15+/-2 fold increase in sCr, Jnk2-/-versus WT, respectively; p=NS). Jnk-PT mice exposed to renal I/R injury showed significant protection from injury compared to WT mice (5.5+/-0.7 vs 15+/-2 fold increase in sCr respectively p<0.01). CC-90001 treatment reduced tubular damage and macrophage infiltration. This protection was replicated in Jnk1-/- and Jnk-PT mice. Conclusion(s): Using complementary ap

Background & Aim: Background: Chronic liver disease is characterised by progressive hepatocyte injury, inflammation and accumulation of extracellular matrix. Liver progenitor cells (LPC) are bipotent cells induced during liver injury that can regenerate both hepatocytes and cholangiocytes. In addition, macrophages play a pivotal role in fibrogenesis and fibrosis resolution. Classically, macrophages are classified into M1 (pro-inflammatory macrophages) and M2 (pro-resolution macrophages). Recently, Ly6C expression has been used to identify distinct macrophage populations, Ly6Chi (inflammatory) and Ly6Clow (resolution). We are developing a therapy for chronic liver fibrosis using human amnion epithelial cells (hAECs). The choline-deficient ethionine-supplemented (CDE) diet is a non-invasive and quick method to induce liver injury and LPC proliferation. Here, we aimed to investigate the effect of hAECs on macrophages and LPC during liver injury induced by a CDE diet. Aim(s): Methods, Results & Conclusion Methods: C57/B16J mice were commenced on a CDE diet, then 2 or 4 million hAECs were infused intraperitoneally 24h later and the CDE diet continued. Outcomes were assessed at days 1, 3, 5, 14, 21 and 42 following hAEC administrations. Sirius red staining and immunohistochemistry were used to determine fibrosis and identify LPC in liver sections. Macrophages and LPC were further identified and characterised using flow cytometery sorting and PCR. Result(s): In CDE treated mice, hAEC significantly reduced liver fibrosis compared to untreated mice. hAEC decreased the number of proinflammatory (Ly6Chi) monocyte-derived macrophages (MDM) and increased the number of anti-inflammatory and tissue remodelling (Ly6Clow) MDM. hAEC also enhanced the expression of matrix-metalloproteinase (MMP)12 and MMP13 in Ly6CLow sorted cells. LPC numbers were reduced significantly in hAEC treated mice. Conclusion(s): Our data confirm the potential efficacy of hAECs as an effective therapy in liv

The importance of tetraspanin proteins in regulating migration has been demonstrated in many diverse cellular systems. However, the function of the leukocyte-restricted tetraspanin CD53 remains obscure. We therefore hypothesized that CD53 plays a role in regulating leukocyte recruitment and tested this hypothesis by examining responses of CD53-deficient mice to a range of inflammatory stimuli. Deletion of CD53 significantly reduced neutrophil recruitment to the acutely inflamed peritoneal cavity. Intravital microscopy revealed that in response to several inflammatory and chemotactic stimuli, absence of CD53 had only minor effects on leukocyte rolling and adhesion in postcapillary venules. In contrast, Cd53-/- mice showed a defect in leukocyte transmigration induced by TNF, CXCL1 and CCL2, and a reduced capacity for leukocyte retention on the endothelial surface under shear flow. Comparison of adhesion molecule expression in wild-type and Cd53-/- neutrophils revealed no alteration in expression of b2 integrins, whereas L-selectin was almost completely absent from Cd53-/- neutrophils. In addition, Cd53-/- neutrophils showed defects in activation-induced cytoskeletal remodeling and translocation to the cell periphery, responses necessary for efficient transendothelial migration, as well as increased a3 integrin expression. These alterations were associated with effects on inflammation, so that in Cd53-/- mice, the onset of neutrophil-dependent serum-induced arthritis was delayed. Together, these findings demonstrate a role for tetraspanin CD53 in promotion of neutrophil transendothelial migration and inflammation, associated with CD53-mediated regulation of L-selectin expression, attachment to the endothelial surface, integrin expression and trafficking, and cytoskeletal function.Copyright © 2020 by The American Association of Immunologists, Inc.

Introduction: The drug treatment for pre-eclampsia-antihy-pertensives-has not changed for 50 years. Targeting the maternal endothelial dysfunction that underlies the clinical disease should ofer improvements in clinical outcomes. Sulforaphane is a potent, naturally occurring antioxidant found in broccoli sprout extract (BSE) that is highly efective at normalising endothelial dysfunction in vitro. To be biologically active, sulforaphane needs to be activated by the enzyme myrosinase. We set out to establish the in vivo pharmacokinetics of an activated and non-activated broccoli sprout formulation in healthy non-pregnant women. Method(s): We performed an open-label, single-arm crossover trial of both BSE preparations. There was a two-week washout period between administration of each preparation. Six healthy women fasted from midnight before ingesting four capsules of myrosinase activated (wave one) or non-activated (wave two) BSE. We collected serum prior to ingestion and at fve time points over eight hours. We quantifed levels of fve sulforaphane metabolites at each time point using liquid chromatography-mass spectrom-etry (LC-MS). We compared the mean area under the curve for each metabolite in activated and non-activated groups. Result(s): The myrosinase activated preparation gave higher levels of sulforaphane-cysteine (444.3 vs. 217.9, p = 0.008), sulforaphane-cysteine-glycine (1555 vs. 870.5, p = 0.03), sulforaphane-glutathione (845.7 vs. 443.9, p = 0.04) and sulforaphane-N-acetyl-cysteine (315.5 VS. 195.5 p = 0.05). There was no diference in non-glycosylated sulforaphane (478 vs 365, p = 0.1). Discussion(s): A myrosinase activated formula of sulforaphane provides signifcantly higher serum levels of key sulforaphane metabolites than a non-activated formula. A pharmacokinetic study of activated sulforaphane in pre-eclamptic women is now worthwhile.

Infection is a leading cause of death in patients with stroke; however, the impact of cerebral infarct size or location on infectious outcome is unclear. To examine the effect of infarct size on post-stroke infection, we utilised the intraluminal middle-cerebral artery occlusion (MCAO) mouse model of ischemic stroke and adjusted the duration of arterial occlusion. At 1 day following stroke onset, the proportion of mice with infection was significantly greater in mice that had larger infarct sizes. Additionally, the presence of lung infection in these mice with severe strokes extended past 2 days, suggestive of long-term immune impairment. At the acute phase, our data demonstrated an inverse relationship between infarct volume and the number of circulating leukocytes, indicating the elevated risk of infection in more severe stroke is associated with reduced cellularity in peripheral blood, owing predominately to markedly decreased lymphocyte numbers. In addition, the stroke-induced reduction of lymphocyte-to-neutrophil ratio was also evident in the lung of all post-stroke animals. To investigate the effect of infarct location on post-stroke infection, we additionally performed a photothrombotic (PT) model of stroke and using an innovative systematic approach of analysis, we found the location of cerebral infarct does not impact on the susceptibility of post-stroke infection, confirming the greater role of infarct volume over infarct location in the susceptibility to infection. Our experimental findings were validated in a clinical setting and reinforced that stroke severity, and not infarct location, influences the risk of infection after stroke.Copyright © 2019, Springer Science+Business Media, LLC, part of Springer Nature.

IgA nephropathy (IgAN), the most common primary glomerular disorder, has a relatively poor prognosis yet lacks a pathogenesisbased treatment. Compound K (CK) is a major absorbable intestinal bacterial metabolite of ginsenosides, which are bioactive components of ginseng. The present study revealed promising therapeutic effects of CK in two complementary IgAN models: a passively induced one developed by repeated injections of IgA immune complexes and a spontaneously occurring model of spontaneous grouped ddY mice. The potential mechanism for CK includes 1) inhibiting the activation of NLRP3 inflammasome in renal tissues, macrophages and bone marrow-derived dendritic cells, 2) enhancing the induction of autophagy through increased SIRT1 expression, and 3) eliciting autophagy-mediated NLRP3 inflammasome inhibition. The results support CK as a drug candidate for IgAN.Copyright © 2020 by The American Association of Immunologists, Inc.

Background: Podocyte depletion is a common feature of glomerulosclerosis (FSGS), but its role in crescentic nephritis remains unclear. This study combined genetic tagging of podocytes with three different optical clearing techniques to determine podocyte depletion in whole glomeruli from mice with crescentic nephritis. Method(s): Podocyte nuclei were labeled by eGFP-histone in of adult male Pod-rtTA/ H2B-eGFP mice by oral doxycycline, followed by a 7-day wash out period, and a single intra-peritoneal injection of nephrotoxic serum (NTS; 5mg/g). Experimental mice were killed 10 days after NTS injection, and compared to age-matched controls. Kidney slices were optically cleared with SCALE-A4, CLARITY and Ethyl Cinnamate (ECi). Highresolution serial optical images were obtained by confocal and two-photon microscopy. Result(s): Mean podocyte number per mouse showed very low variability within controls (1-5% variability, P>0.05) and within NTS-injected mice (1-9% variability, P>0.05) independent of the clearing technique. In NTS-injected mice, a similar degree of average podocyte depletion per mouse was identified with all clearing methods (60-63%, P<0.001). The technical (dis-)advantages of each clearing protocol were also analysed, including optimal penetration depth and resolution, compatibility with immunofluorescence, microscopy set-ups, and cost-efficiency. Importantly, total podocyte number per glomerulus showed great variability: controls (mean: 78,81; ranging from 49 to 128 podocytes per glomerulus) and in NTS-injected mice (mean: 29,99; ranging from 1 to 95 podocytes per glomerulus). Using the lowest value for podocyte number in controls as a cut-off reference, only 78% of analysed glomeruli (141 of 180) from NTS-injected mice had a certain degree of podocyte depletion. While deposition of the NTS within glomeruli occurred in a global and homogeneous fashion, podocyte loss was focal. Conclusion(s): This study has identified early focal podocyte depletion in mic

Although it is well established that microbial infections predispose to autoimmune diseases, the underlying mechanisms remain poorly understood. After infection, tissue-resident memory T (TRM) cells persist in peripheral organs and provide immune protection against reinfection. However, whether TRM cells participate in responses unrelated to the primary infection, such as autoimmune inflammation, is unknown. By using high-dimensional single-cell analysis, we identified CD4+ TRM cells with a TH17 signature (termed TRM17 cells) in kidneys of patients with ANCA-associated glomerulonephritis. Experimental models demonstrated that renal TRM17 cells were induced by pathogens infecting the kidney, such as Staphylococcus aureus, Candida albicans, and uropathogenic Escherichia coli, and persisted after the clearance of infections. Upon induction of experimental glomerulonephritis, these kidney TRM17 cells rapidly responded to local proinflammatory cytokines by producing IL-17A and thereby exacerbate renal pathology. Thus, our data show that pathogen-induced TRM17 cells have a previously unrecognized function in aggravating autoimmune disease.Copyright © 2020 American Association for the Advancement of Science. All rights reserved.

Cyclophilin A (CypA) is a highly abundant protein in the cytoplasm of most mammalian cells. Beyond its homeostatic role in protein folding, CypA is a Damage-Associated Molecular Pattern which can promote inflammation during tissue injury. However, the role of CypA in kidney disease is largely unknown. This study investigates the contribution of CypA in two different types of kidney injury: acute tubular necrosis and progressive interstitial fibrosis. CypA (Ppia) gene deficient and wild type (WT) littermate controls underwent bilateral renal ischaemia/reperfusion injury (IRI) and were killed 24h later or underwent left unilateral ureteric obstruction (UUO) and were killed 7 days later. In the IRI model, CypA-/- mice showed substantial protection against the loss of renal function and from tubular cell damage and death. This was attributed to a significant reduction in neutrophil and macrophage infiltration since CypA-/- tubular cells were not protected from oxidant-induced cell death in vitro. In the UUO model, CypA-/- mice were not protected from leukocyte infiltration or renal interstitial fibrosis. In conclusion, CypA promotes inflammation and acute kidney injury in renal IRI, but does not contribute to inflammation or interstitial fibrosis in a model of progressive kidney fibrosis.Copyright © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

Brain-derived neurotrophic factor (BDNF) has been implicated in cognition and the effects of chronic stress. We have previously shown in mice that chronic adolescent treatment with corticosterone (CORT), to simulate stress, resulted in spatial memory deficits and markedly elevated levels of the N-methyl-D-aspartate (NMDA) receptor subunit NR2B in adult male BDNF heterozygous mice (BDNF+/-), but not in wildtype controls (WT) or females. The aim of the present study was to further characterize this 'two hit' model, including whether these effects are long-lasting. CORT treatment was delivered in the drinking water from 6 to 9 weeks of age. As previously demonstrated, male BDNF+/- mice treated with CORT presented with a deficit in spatial memory at 11 weeks of age. However, this deficit was not maintained at 15 weeks of age. Conversely, male WT treated with CORT developed a deficit only at 15 weeks of age. There were no significant gene-environment interactions in female mice at any time point. CORT treatment caused a modest, but significant increase in NR2B levels which was independent of genotype. These results show marked age-dependent and sex-dependent effects of CORT on behaviour which are different in BDNF+/- mice than in controls.Copyright © 2019 Elsevier B.V.

Detailed morphological characterization of testicular leukocytes in the adult CX3CR1 gfp/+ transgenic mouse identified two distinct CX3CR1 + mononuclear phagocyte (macrophage and dendritic cell) populations: stellate/dendriform cells opposed to the seminiferous tubules (peritubular), and polygonal cells associated with Leydig cells (interstitial). Using confocal microscopy combined with stereological enumeration of CX3CR1 gfp/+ cells established that there were twice as many interstitial cells (68%) as peritubular cells (32%). Flow cytometric analyses of interstitial cells from mechanically-dissociated testes identified multiple mononuclear phagocyte subsets based on surface marker expression (CX3CR1, F4/80, CD11c). These cells comprised 80% of total intratesticular leukocytes, as identified by CD45 expression. The remaining leukocytes were CD3+ (T lymphocytes) and NK1.1+ (natural killer cells). Functional phenotype assessment using CD206 (an anti-inflammatory/M2 marker) and MHC class II (an activation marker) identified a potentially tolerogenic CD206+MHCII+ sub-population (12% of total CD45+ cells). Rare testicular subsets of CX3CR1 +CD11c+F4/80+ (4.3%) mononuclear phagocytes and CD3+NK1.1+ (3.1%) lymphocytes were also identified for the first time. In order to examine the potential for the immunoregulatory cytokine, activin A to modulate testicular immune cell populations, testes from adult mice with reduced activin A (Inhba+/-) or elevated activin A (Inha+/-) were assessed using flow cytometry. Although the proportion of F4/80+CD11b+ leukocytes (macrophages) was not affected, the frequency of CD206+MHCII+cells was significantly lower and CD206+MHCII- correspondingly higher in Inha+/- testes. This shift in expression of MHCII in CD206+ macrophages indicates that changes in circulating and/or local activin A influence resident macrophage activation and phenotype and, therefore, the immunological environment of the testis.Copyright © 2020

Arterial thrombosis causes heart attacks and most strokes and is the most common cause of death in the world. Platelets are the cells that form arterial thrombi, and antiplatelet drugs are the mainstay of heart attack and stroke prevention. Yet, current drugs have limited efficacy, preventing fewer than 25% of lethal cardiovascular events without clinically relevant effects on bleeding. The key limitation on the ability of all current drugs to impair thrombosis without causing bleeding is that they block global platelet activation, thereby indiscriminately preventing platelet function in hemostasis and thrombosis. Here, we identify an approach with the potential to overcome this limitation by preventing platelet function independently of canonical platelet activation and in a manner that appears specifically relevant in the setting of thrombosis. Genetic or pharmacological targeting of the class II phosphoinositide 3-kinase (PI3KC2alpha) dilates the internal membrane reserve of platelets but does not affect activation-dependent platelet function in standard tests. Despite this, inhibition of PI3KC2alpha is potently antithrombotic in human blood ex vivo and mice in vivo and does not affect hemostasis. Mechanistic studies reveal this antithrombotic effect to be the result of impaired platelet adhesion driven by pronounced hemodynamic shear stress gradients. These findings demonstrate an important role for PI3KC2alpha in regulating platelet structure and function via a membrane-dependent mechanism and suggest that drugs targeting the platelet internal membrane may be a suitable approach for antithrombotic therapies with an improved therapeutic window. c 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.Copyright © 2020 American Association for the Advancement of Science. All rights reserved.

Background: Cyclophilin D (CypD) facilitates mitochondrial-dependant cell death during pathological conditions. CypD-/-mice are protected from acute kidney injury (AKI) following ischaemia/reperfusion injury and show reduced renal fibrosis in the unilateral ureteric obstruction model. However, the contribution of CypD in aristolochic acid (AA) induced AKI or AA-induced chronic kidney disease (CKD) is unknown. We aim to determine the role of CypD in: 1) acute AA-induced nephropathy (AAN); and 2) chronic AAN. Method(s): Groups (n=10) of CypD-/-and wild type (WT) C57BL/6J mice were used. Study 1: Mice were given an intraperitoneal (IP) injection of 5mg/kg AA and killed 3 days later. Study 2: Mice were given IP injections of 2mg/kg AA every 2nd day and killed on day 28. Controls were untreated. Result(s): Study 1: Acute high dose AA caused renal failure in WT mice (39.7+/-7.1mmol/L vs 13.5+/-2.3mmol/L serum creatinine (SCr) in controls; P<0.0001) with evidence of tubular damage and cell death on PAS sections and increased cleaved caspase-3+ cells. Acute AAN also caused inflammation with infiltrating neutrophils and T cells and up-regulation of IL-36a mRNA levels. CypD-/-mice were protected from AA-induced acute renal dysfunction (18.9+/-9.5 mmol/L SCr; P<0.0001 vs WT AAN). CypD-/-mice showed reduced tubular damage and cell death on PAS and reduced cleaved caspase-3+ cells (both P<0.001 vs WT AAN), as well as reduced neutrophil infiltration and IL-36alpha mRNA levels (both P<0.001 vs WT AAN). Study 2: Chronic AA administration caused renal impairment in WT mice (34.3+/-9.9mmol/L SCr), with evidence of chronic tubular damage (KIM-1 & a-Klotho mRNA levels), increase in tubular cell death (cleaved caspase-3+ cells), and significant renal fibrosis (increased collagen IV deposition). However, CypD-/-mice were not protected from chronic AA-induced renal dysfunction (37.0+/-14.3 mmol/L SCr; P=NS) and showed no reduction in tubular damage, cell death or renal fibrosis. Conclusio

Unresolved inflammation can lead to tissue fibrosis and impaired organ function. Macrophage-myofibroblast transition (MMT) is one newly identified mechanism by which ongoing chronic inflammation causes progressive fibrosis in different forms of kidney disease. However, the mechanisms underlying MMT are still largely unknown. Here, we discovered a brain-specific homeobox/POU domain protein Pou4f1 (Brn3a) as a specific regulator of MMT. Interestingly, we found that Pou4f1 is highly expressed by macrophages undergoing MMT in sites of fibrosis in human and experimental kidney disease, identified by coexpression of the myofibroblast marker, alpha-SMA. Unexpectedly, Pou4f1 expression peaked in the early stage in renal fibrogenesis in vivo and during MMT of bone marrow-derived macrophages (BMDMs) in vitro. Mechanistically, chromatin immunoprecipitation (ChIP) assay identified that Pou4f1 is a Smad3 target and the key downstream regulator of MMT, while microarray analysis defined a Pou4f1-dependent fibrogenic gene network for promoting TGFbeta1/Smad3-driven MMT in BMDMs at the transcriptional level. More importantly, using two mouse models of progressive renal interstitial fibrosis featuring the MMT process, we demonstrated that adoptive transfer of TGF-beta1-stimulated BMDMs restored both MMT and renal fibrosis in macrophage-depleted mice, which was prevented by silencing Pou4f1 in transferred BMDMs. These findings establish a role for Pou4f1 in MMT and renal fibrosis and suggest that Pou4f1 may be a therapeutic target for chronic kidney disease with progressive renal fibrosis.Copyright © 2020 National Academy of Sciences. All rights reserved.

Background: Cyclophilins are a family of enzymes which regulate protein folding. Of these enzymes, cyclophilin D (CypD) facilitates mitochondrial-dependant cell death during pathological conditions. CypD-/-mice are protected from acute kidney injury (AKI) following renal ischaemia/reperfusion injury, and exhibit less renal fibrosis in the unilateral ureteric obstruction model. However, the contribution of CypD in the transition of AKI to chronic kidney disease (CKD) is not known. The aim of this study was to determine the role of CypD in promoting the transition of AKI to CKD using the aristolochic acid-induced nephropathy (AAN) model. Method(s): Groups (n=10) of wild type (WT) and CypD-/-mice on the C57BL/6J background were given intraperitoneal injections of 2mg/kg aristolochic acid every 2nd day for 28 days. Mice were killed on day 28. Controls were untreated. Result(s): AAN caused >2.5-fold rise in serum creatinine (sCr) in WT mice (34.3+/-9.9mmol/L vs 13.5+/-2.3mmol/L in controls; P<0.0001), with evidence of tubular damage (KIM-1 & alpha-Klotho mRNA levels; P<0.0001), increase in tubular cell death (cleaved caspase-3+ cells; P<0.0001), and significant renal fibrosis (collagen IV immunostaining, and collagen I & alpha-SMA mRNA levels; all P<0.0001 vs controls). CypD-/-mice were not protected from AA-induced renal dysfunction (sCr 37.0+/-14.3 mmol/L; P=N.S.), and showed no reduction in tubular damage, cell death or renal fibrosis. Further analysis showed that AAN in WT mice caused loss of peritubular CD31+ capillaries (P<0.0001), and infiltration of macrophages (CD68 & CD206 mRNA levels; P<0.0001) and T-cells (CD3 & IL-2 mRNA levels; P<0.0001). CypD-/-mice were not protected from peritubular capillary loss or macrophage infiltration in AAN. However, CypD-/-mice showed reduced T-cell infiltration and activation (CD3 & IL-2 mRNA levels; P<0.0001), including Th1 cells (T-bet mRNA levels; P<0.05). Conclusion(s): CypD does not contribute to the transition of AKI to CK

Kidney fibrosis is characterized by expansion and activation of platelet-derived growth factor receptor-beta (PDGFR-beta)-positive mesenchymal cells. To study the consequences of PDGFR-beta activation, we developed a model of primary renal fibrosis using transgenic mice with PDGFR-beta activation specifically in renal mesenchymal cells, driving their pathological proliferation and phenotypic switch toward myofibroblasts. This resulted in progressive mesangioproliferative glomerulonephritis, mesangial sclerosis, and interstitial fibrosis with progressive anemia due to loss of erythropoietin production by fibroblasts. Fibrosis induced secondary tubular epithelial injury at later stages, coinciding with microinflammation, and aggravated the progression of hypertensive and obstructive nephropathy. Inhibition of PDGFR activation reversed fibrosis more effectively in the tubulointerstitium compared to glomeruli. Gene expression signatures in mice with PDGFR-beta activation resembled those found in patients. In conclusion, PDGFR-beta activation alone is sufficient to induce progressive renal fibrosis and failure, mimicking key aspects of chronic kidney disease in humans. Our data provide direct proof that fibrosis per se can drive chronic organ damage and establish a model of primary fibrosis allowing specific studies targeting fibrosis progression and regression.Copyright © 2020 The Authors. Published under the terms of the CC BY 4.0 license

Background: Neonatal ventilation exacerbates brain injury in lambs with fetal growth restriction (FGR), characterized by neuroinflammation and reduced blood-brain barrier integrity, which is normally maintained by the neurovascular unit. We examined whether umbilical cord blood stem cell (UCBC) treatment stabilized the neurovascular unit and reduced brain injury in preterm ventilated FGR lambs. Method(s): Surgery was performed in twin-bearing pregnant ewes at 88 days' gestation to induce FGR in one fetus. At 127 days, FGR and appropriate for gestational age (AGA) lambs were delivered, carotid artery flow probes and umbilical lines inserted, lambs intubated and commenced on gentle ventilation. Allogeneic ovine UCBCs (25 x 106 cells/kg) were administered intravenously to lambs at 1 h of life. Lambs were ventilated for 24 h and then euthanized. Result(s): FGR (n = 6) and FGR+UCBC (n = 6) lambs were growth restricted compared to AGA (n = 6) and AGA+UCBC (n = 6) lambs (combined weight, FGR 2.3 +/- 0.4 vs. AGA 3.0 +/- 0.3 kg; p = 0.0002). UCBC therapy did not alter mean arterial blood pressure or carotid blood flow but decreased cerebrovascular resistance in FGR+UCBC lambs. Circulating TNF-alpha cytokine levels were lower in FGR+UCBC vs. FGR lambs (p < 0.05). Brain histopathology showed decreased neuroinflammation and oxidative stress, increased endothelial cell proliferation, pericyte stability, and greater integrity of the neurovascular unit in FGR+UCBC vs. FGR lambs. Conclusion(s): Umbilical cord blood stem cell therapy mitigates perinatal brain injury due to FGR and ventilation, and the neuroprotective benefits may be mediated by stabilization of the neurovascular unit.Copyright © 2020 The Author(s).

Dopamine is often used to treat hypotension in preterm infants who are at risk of hypoxic-ischemic (HI) brain injury due to cerebral hypoperfusion and impaired autoregulation. There is evidence that systemically administered dopamine crosses the preterm blood-brain barrier. However, the effects of exogenous dopamine and cerebral HI on dopaminergic signaling in the immature brain are unknown. We determined the effect of HI and dopamine on D1 and D2 receptor binding and expressions of dopamine transporter (DAT) and tyrosine hydroxylase (TH) in the striatum of the preterm fetal sheep. Fetal sheep (99 days of gestation, term=147days) were unoperated controls (n = 6) or exposed to severe HI using umbilical cord occlusion and saline infusion (UCO + saline, n = 8) or to HI with dopamine infusion (UCO + dopamine, 10 microg/kg/min, n = 7) for 74 h. D1 and D2 receptor densities were measured by autoradiography in vitro. DAT, TH, and cell death were measured using immunohistochemistry. HI resulted in cell death in the caudate nucleus and putamen, and dopamine infusion started before HI did not exacerbate or ameliorate these effects. HI led to reduced D1 and D2 receptor densities in the caudate nucleus and reduction in DAT protein expression in the caudate and putamen. Fetal brains exposed to dopamine in addition to HI were not different from those exposed to HI alone in these changes in dopaminergic parameters. We conclude that dopamine infusion does not alter the striatal cell death or the reductions in D1 and D2 receptor densities and DAT protein expression induced by HI in the preterm brain.NEW & NOTEWORTHY This is the first study on the effects of hypoxia-ischemia and dopamine treatment on the dopaminergic pathway in the preterm brain. In the striatum of fetal sheep (equivalent to ~26-28 wk of human gestation), we demonstrate that hypoxia-ischemia leads to cell death, reduces D1 and D2 receptors, and reduces dopamine transporter. Intravenous dopamine infusion at clinical d

MHC class II (MHC II) displays peptides at the cell surface, a process critical for CD4+ T cell development and priming. Ubiquitination is a mechanism that dictates surface MHC II with the attachment of a polyubiquitin chain to peptide-loaded MHC II, promoting its traffic away from the plasma membrane. In this study, we have examined how MHC II ubiquitination impacts the composition and function of both conventional CD4+ T cell and regulatory T cell (Treg) compartments. Responses were examined in two models of altered MHC II ubiquitination: MHCIIKRKI/KI mice that express a mutant MHC II unable to be ubiquitinated or mice that lack membrane-associated RING-CH 8 (MARCH8), the E3 ubiquitin ligase responsible for MHC II ubiquitination specifically in thymic epithelial cells. Conventional CD4+ T cell populations in thymus, blood, and spleen of MHCIIKRKI/KI and March82/2 mice were largely unaltered. In MLRs, March82/2, but not MHCIIKRKI/KI, CD4+ T cells had reduced reactivity to both self- A nd allogeneic MHC II. Thymic Treg were significantly reduced in MHCIIKRKI/KI mice, but not March82/2 mice, whereas splenic Treg were unaffected. Neither scenario provoked autoimmunity, with no evidence of immunohistopathology and normal levels of autoantibody. In summary, MHC II ubiquitination in specific APC types does not have a major impact on the conventional CD4+ T cell compartment but is important for Treg development. The Journal of Immunology, 2020, 205: 1207-1216.Copyright © 2020 by The American Association of Immunologists, Inc.

With approximately 131 million new genital tract infections occurring each year, Chlamydia is the most common sexually transmitted bacterial pathogen worldwide. Male and female infections occur at similar rates and both cause serious pathological sequelae. Despite this, the impact of chlamydial infection on male fertility has long been debated, and the effects of paternal chlamydial infection on offspring development are unknown. Using a male mouse chronic infection model, we show that chlamydial infection persists in the testes, adversely affecting the testicular environment. Infection increased leukocyte infiltration, disrupted the blood:testis barrier and reduced spermiogenic cell numbers and seminiferous tubule volume. Sperm from infected mice had decreased motility, increased abnormal morphology, decreased zona-binding capacity, and increased DNA damage. Serum anti-sperm antibodies were also increased. When both acutely and chronically infected male mice were bred with healthy female mice, 16.7% of pups displayed developmental abnormalities. Female offspring of chronically infected sires had smaller reproductive tracts than offspring of noninfected sires. The male pups of infected sires displayed delayed testicular development, with abnormalities in sperm vitality, motility, and sperm-oocyte binding evident at sexual maturity. These data suggest that chronic testicular Chlamydia infection can contribute to male infertility, which may have an intergenerational impact on sperm quality.Copyright © 2020 The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction.

Background: Optical clearing and advanced light microscopy have revolutionised three-dimensional quantification in cell biology. Here, we present a Systematic Pipeline of Enhanced Clarification for Three-dimensional Rendering and Unbiased Morphometrics (SPECTRUM) as a unique tool for the comprehensive analysis of glomerular health and disease. Method(s): SPECTRUM is based on a combination of single cell identification (ie. lineage tracing), optical clearing, advanced light microscopy with single cell resolution, and 3D morphometrics. Podocytes and parietal epithelial cells (PECs) were genetically labelled with eGFP using inducible mouse systems (POD or PEC -rtTA/H2B-eGFP). Crescentic nephritis was used as a validation model. Several optical clearing protocols were optimized for kidney tissue, including aqueous, hydrogel, and solvent-based approaches. Image acquisition was based on whole structures (ie. glomeruli and lesions) using light sheet, confocal and two-photon microscopy. Subsequent tissue de-clarification with immunolabelling and classical histopathology allowed the combination of 3D and 2D analyses. Result(s): While IgG deposition showed a homogeneous distribution among all analysed glomeruli, only 80 showed signs of podocyte loss (podocyte loss per glomerulus of about 60). Only glomerular lesions showed significant increases in numbers of PECs (sign of PEC activation) in close association with reductions in glomerular capillary volume (sign of capillary injury). PEC activation was confirmed via immunofluorescence in recycled tissue samples (ie. de-novo CD44 expression). Based on 3D analysis of intraglomerular lesion location, we identified a progressive pattern from localised tubular lesions to segmental lesions and development of atubular glomeruli. Conclusion(s): SPECTRUM provides new roadmap for morphometrics in the kidney. In crescentic nephritis, SPECTRUM revealed that despite of uniform IgG deposition, there was focal lesion development and podocyte

Glucocorticoid (GC) signaling via the glucocorticoid receptor (GR) is essential for lung maturation in mammals. Previous studies using global or conditional mouse model knockouts of the GR gene have established that GR-mediated signaling in the interstitial mesenchyme of the fetal lung is critical for normal lung development. Screens for downstream GC-targets in conditional mesenchymal GR deficient mouse lung (GRmesKO) identified Versican (Vcan), an important extracellular matrix component and cell proliferation regulator, as a potential GR-regulated target. We show that, of the five major VCAN isoforms, the VCAN-V1 isoform containing the GAGbeta domain is the predominant VCAN isoform in the fetal mouse lung distal mesenchyme at both E16.5 and E18.5, whereas the GAGalpha-specific VCAN-V2 isoform was only localized to the smooth muscle surrounding proximal airways. Both Vcan-V1 mRNA and protein levels were strongly overexpressed in the GRmesKO lung at E18.5. Finally, we investigated the GC regulation of the ECM protease ADAMTS 12 and showed that Adamts 12 mRNA levels were markedly reduced at E18.5 in GRmesKO fetal mouse lung and were strongly induced by both cortisol and betamethasone in cultures of primary rat fetal lung fibroblasts. ADAMTS12 protein immunoreactivity was also strongly increased in the distal lung at E18.5, after dexamethasone treatment in utero. In summary, glucocorticoid signaling via GR represses GAGbeta domain-containing VCAN isoforms in distal lung mesenchyme in vivo by repressing Vcan gene expression and, in part, by inducing the ECM protease ADAMTS12, thereby contributing to the control of ECM remodelling and lung cell proliferation prior to birth.Copyright © 2020 Society for Endocrinology Published by Bioscientifica Ltd.

MR activation in macrophages is critical for the development of cardiac inflammation and fibrosis. We previously showed that MR activation modifies macrophage pro-inflammatory signalling, changing the cardiac tissue response to injury via both direct gene transcription and JNK/AP-1 second messenger pathways. In contrast, MR-mediated renal electrolyte homeostasis is critically determined by DNA-binding-dependent processes. Hence, ascertaining the relative contribution of MR actions via DNA binding or alternative pathways on macrophage behaviour and cardiac inflammation may provide therapeutic opportunities which separate the cardioprotective effects of MR antagonists from their undesirable renal potassium-conserving effects. We developed new macrophage cell lines either lacking MR or harbouring a mutant MR incapable of DNA binding. Western blot analysis demonstrated that MR DNA binding is required for lipopolysaccharide (LPS), but not phorbol 12-myristate-13-acetate (PMA), induction of the MAPK/pJNK pathway in macrophages. Quantitative RTPCR for pro-inflammatory and pro-fibrotic targets revealed subsets of LPS- and PMA-induced genes that were either enhanced or repressed by the MR via actions that do not always require direct MR-DNA binding. Analysis of the MR target gene and profibrotic factor MMP12 identified promoter elements that are regulated by combined MR/MAPK/JNK signalling. Evaluation of cardiac tissue responses to an 8-day DOC/salt challenge in mice selectively lacking MR DNA-binding in macrophages demonstrated levels of inflammatory markers equivalent to WT, indicating non-DNA binding-dependent MR signalling in macrophages is sufficient for DOC/salt-induced tissue inflammation. Our data demonstrate that the MR regulates a macrophage pro-inflammatory phenotype and cardiac tissue inflammation, partially via pathways that do not require DNA binding.Copyright © 2020 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain

Background: Cyclophilins are enzymes that regulate protein folding. During various pathological conditions, cyclophilin A (CypA) has been shown to promote leukocyte recruitment and inflammation. However, the contribution of CypA in acute kidney injury (AKI) and renal fibrosis is poorly understood. The aim of this study was to determine the role of CypA in renal tubular damage, inflammation, and fibrosis using the: (1) renal ischaemia/reperfusion injury (IRI), and (2) unilateral ureteric obstruction (UUO) models. Method(s): Groups (n=10) of wild type (WT) and CypA-/-mice on the 129 background were subjected to either: (1) bilateral renal IRI (19min at 37degreeC) and killed 24 hours post reperfusion, or (2) UUO surgery and killed on day 7. Controls were sham operated. Result(s): Renal IRI induced AKI with a >6-fold rise in serum creatinine (sCr) in WT mice (41+/-13.8 vs 6.3+/-1.7umol/L in sham; P<0.0001) and marked tubular damage based on the tubular injury score, increased KIM-1 mRNA levels, and tubular cell death (TUNEL+ cells; all P<0.0001 vs sham). CypA-/-mice were protected from renal dysfunction with a 50% improvement of sCr (20.7+/-3.5umol/L; P<0.0001), less histologic tubular damage (P<0.01), lower KIM-1 mRNA levels (P<0.01), and less tubular cell death (P<0.001). Renal IRI caused upregulated mRNA levels of inflammatory molecules (TNF-alpha and IL-36-alpha; P<0.05), and neutrophil infiltration (Ly6G+ cells; P<0.0001). CypA-/-mice had a clear reduction in inflammatory molecules (P<0.05) and less infiltrating neutrophils (P<0.001). UUO in WT mice resulted in significant renal fibrosis (3-fold increase in collagen IV deposition by immunostaining; P<0.001 vs sham), with significant infiltrates of macrophages (F4/80+ cells), T cells (CD3+ cells) and neutrophils (Ly6G+ cells) (all P<0.0001 vs sham). CypA-/-mice were not protected from renal fibrosis or leukocyte infiltration in the UUO model. Conclusion(s): CypA contributes to leukocyte infiltration and inflammation

Background: Neutrophil extracellular trap (NETs) are implicated in the pathogenesis of myeloperoxidase anti neutrophil cytoplasmic antibody vasculitis (MPO-AAV). Neutrophil elastase (NE) is implicated in initiating NET formation through proteolytic degradation of the nuclear envelope, histone degradation and chromatin decondensation in concert with other neutrophil enzymes to facilitate DNA release. NETs are decorated with multiple proinflammatory enzymes including MPO (the autoantigen in this disease) and NE. NE released through degranulation is degraded by endogenous NE inhibitors, however NE bound to NET derived DNA is protected providing a reservoir of proteolytic NE enabling tissue destruction, microvascular injury and further recruitment of neutrophils. This study investigates the pathogenic contribution of NE release through NET formation in experimental MPO-AAV using animals deficient in neutrophil elastase. Method(s): A 20 day experimental model of anti-MPO GN was induced in B6.129X1-Elanetm1Sds/J mice (neutrophil elastase knock out mice) and wild-type (WT) litter mate controls by myeloperoxidase (MPO) immunisation and GN triggered using a subnephritogenic dose of anti-glomerular basement membrane globulin (anti GBM Ig), [n=10 (WT) and n=10 (Elane-/-)]. Result(s): Elane-/-animals were protected from excessive NET production and glomerular injury with significantly reduced numbers of glomerular neutrophils, extracellular MPO deposition, CD4 T cells and macrophages influx (all, P <0.05 compared to WT). Histological assessment of kidneys demonstrated a reduction in segmental necrosis in the Elane-/-group compared to the WT group (P<0.05). Functional injury measured by creatinine/albuminuria ratio was significantly reduced in the Elane -/-group compared to the WT group (P=0.04). Systemic autoimmunity to MPO was significantly reduced in Elane-/-mice with reduced frequency of MPO specific CD4 effector T cells from the draining lymph nodes and a significant reduct

Background: The reactive oxygen species-inducible JNK signaling pathway is activated in tubular cells within 20min of renal ischaemia/reperfusion (I/R) injury. Tubular cell death following I/R injury is reduced by systemic JNK inhibitor treatment, but whether this is a direct or indirect effect and the contribution of JNK1 versus JNK2 isoforms are unclear. We aimed to define the role of JNK1 versus JNK2 signaling in oxidant-induced tubular cell death and whether this mechanism also operates in diabetes. Method(s): Bilateral renal I/R injury was induced in groups (n=8 to 10) of male wild type (WT), Jnk1-/-, Jnk2-/-, and Jnk1f/f gammaGT-Cre mice (all C57BL/6J) which were killed 24hr later. Diabetes was induced in WT mice by 5 low dose streptozotocin (STZ) injections. Result(s): Compared to renal I/R injury in WT mice, Jnk1-/-but not Jnk2-/-mice were significantly protected from tubular cell death (PAS and cleaved caspase 3 staining) and renal failure (serum creatinine) (both P<0.01). Furthermore, conditional deletion of Jnk1 in proximal tubular cells (Jnk1f/f gammaGT-Cre mice) also showed a significant reduction in tubular cell death and renal failure (both P<0.01 vs WT I/R). H2O2 (0.25 to 1mM) induced dose-dependent cell death in primary cultures of WT tubular epithelial cells (TEC); however, Jnk1-/-TEC showed 50% cell death (P<0.001), whereas Jnk2-/-TEC showed no protection. A JNK inhibitor reduced H2O2-induced cell death in WT and Jnk2-/-TEC by 50%, but did not affect cell death in Jnk1-/-TEC. In a further study, renal I/R injury was induced in WT mice 8 weeks after STZ-induced diabetes. Compared to non-diabetic controls, I/R injury caused increased JNK signalling, greater tubular cell death and more severe renal failure (all P<0.01 vs non-diabetic I/R). Culture of WT TEC in high glucose for 48hr increased H2O2-induced cell death. However, a JNK inhibitor reduced H2O2-induced cell death under high glucose by 38 to 45% (P<0.001). Conclusion(s): JNK1 but not JNK2 dir

Rationale: Atypical dopamine (DA) transport blockers such as modafinil and its analogs may be useful for treating motivational symptoms of depression and other disorders. Previous research has shown that the DA depleting agent tetrabenazine can reliably induce motivational deficits in rats, as evidenced by a shift towards a low-effort bias in effort-based choice tasks. This is consistent with human studies showing that people with major depression show a bias towards low-effort activities. Objectives: Recent studies demonstrated that the atypical DA transport (DAT) inhibitor (S)-CE-123 reversed tetrabenazine-induced motivational deficits, increased progressive ratio (PROG) lever pressing, and increased extracellular DA in the nucleus accumbens. In the present studies, a recently synthesized modafinil analog, (S, S)-CE-158, was assessed in a series of neurochemical and behavioral studies in rats. Results: (S, S)-CE-158 demonstrated the ability to reverse the effort-related effects of tetrabenazine and increase selection of high-effort PROG lever pressing in rats tested on PROG/chow feeding choice task. (S, S)-CE-158 showed a high selectivity for inhibiting DAT compared with other monoamine transporters, and systemic administration of (S, S)-CE-158 increased extracellular DA in the nucleus accumbens during the behaviorally active time course, which is consistent with the effects of (S)-CE-123 and other DAT inhibitors that enhance high-effort responding. Conclusions: These studies provide an initial neurochemical characterization of a novel atypical DAT inhibitor, and demonstrate that this compound is active in models of effort-related choice. This research could contribute to the development of novel compounds for the treatment of motivational dysfunctions in humans. © 2020, Springer-Verlag GmbH Germany, part of Springer Nature.

Neuromuscular interfaces are required to translate bioelectronic technologies for application in clinical medicine. Here, by leveraging the robotically controlled ink-jet deposition of low-viscosity conductive inks, extrusion of insulating silicone pastes and in situ activation of electrode surfaces via cold-air plasma, we show that soft biocompatible materials can be rapidly printed for the on-demand prototyping of customized electrode arrays well adjusted to specific anatomical environments, functions and experimental models. We also show, with the monitoring and activation of neuronal pathways in the brain, spinal cord and neuromuscular system of cats, rats and zebrafish, that the printed bioelectronic interfaces allow for long-term integration and functional stability. This technology might enable personalized bioelectronics for neuroprosthetic applications. © 2020, The Author(s), under exclusive licence to Springer Nature Limited.

Traumatic brain injury (TBI) often causes massive brain cell death accompanied by the accumulation of toxic factors in interstitial and cerebrospinal fluids. The persistence of the damaged brain area is not transient and may occur within days and weeks. Chaperone Hsp70 is known for its cytoprotective and antiapoptotic activity, and thus, a therapeutic approach based on chemically induced Hsp70 expression may become a promising approach to lower post-traumatic complications. To simulate the processes of secondary damage, we used an animal model of TBI and a cell model based on the cultivation of target cells in the presence of cerebrospinal fluid (CSF) from injured rats. Here we present a novel low molecular weight substance, PQ-29, which induces the synthesis of Hsp70 and empowers the resistance of rat C6 glioma cells to the cytotoxic effect of rat cerebrospinal fluid taken from rats subjected to TBI. In an animal model of TBI, PQ-29 elevated the Hsp70 level in brain cells and significantly slowed the process of the apoptosis in acceptor cells in response to cerebrospinal fluid action. The compound was also shown to rescue the motor function of traumatized rats, thus proving its potential application in rehabilitation therapy after TBI. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

Aims: Mutations in DNA/RNA-binding factor (fused-in-sarcoma) FUS and superoxide dismutase-1 (SOD-1) cause amyotrophic lateral sclerosis (ALS). They were reproduced in SOD-1-G93A (SOD-1) and new FUS[1-359]-transgenic (FUS-tg) mice, where inflammation contributes to disease progression. The effects of standard disease therapy and anti-inflammatory treatments were investigated using these mutants. Methods: FUS-tg mice or controls received either vehicle, or standard ALS treatment riluzole (8 mg/kg/day), or anti-inflammatory drug a selective blocker of cyclooxygenase-2 celecoxib (30 mg/kg/day) for six weeks, or a single intracerebroventricular (i.c.v.) infusion of Neuro-Cells (a preparation of 1.39 × 106 mesenchymal and hemopoietic human stem cells, containing 5 × 105 of CD34+ cells), which showed anti-inflammatory properties. SOD-1 mice received i.c.v.-administration of Neuro-Cells or vehicle. Results: All FUS-tg-treated animals displayed less marked reductions in weight gain, food/water intake, and motor deficits than FUS-tg-vehicle-treated mice. Neuro-Cell-treated mutants had reduced muscle atrophy and lumbar motor neuron degeneration. This group but not celecoxib-FUS-tg-treated mice had ameliorated motor performance and lumbar expression of microglial activation marker, ionized calcium-binding adapter molecule-1 (Iba-1), and glycogen-synthase-kinase-3ß (GSK-3ß). The Neuro-Cells-treated-SOD-1 mice showed better motor functions than vehicle-treated-SOD-1 group. Conclusion: The neuropathology in FUS-tg mice is sensitive to standard ALS treatments and Neuro-Cells infusion. The latter improves motor outcomes in two ALS models possibly by suppressing microglial activation. © 2019 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd