We have studied endothelin-1 (ET-1) levels and ET-1 ligand and receptor tissue expression in scleroderma renal crisis (SRC) and undertaken a pilot open label safety study of bosentan, a non-selective ...ET-1 receptor antagonist, in SRC Bosentan in Renal Disease-1 (BIRD-1).
Serum levels of ET-1 were measured in healthy controls (n = 20) or systemic sclerosis (SSc) (n = 80) with or without SRC, including cases of pulmonary arterial hypertension (PAH). Renal biopsies (n = 27) from patients with SRC were stained for endothelin ligand and receptors. Six cases of SRC received 6 months bosentan. Outcome measures were compared with SRC cases managed at our centre from 2000 to 2004 (n = 49).
Serum ET-1 was elevated in SRC but less than in PAH. ET-1 and both endothelin A and endothelin B receptor expression was increased in SRC biopsies in glomeruli, interstitium and hallmark vascular lesions of SRC. In the BIRD-1 cohort, serum ET-1 was elevated in all cases at SRC (median healthy controls 0.50 pg/ml; SRC 1.48 pg/ml; P < 0.0005), and increased further with bosentan therapy (1.46 vs. 3.05 pg/ml; t-test P < 0.05). Bosentan was well tolerated with no significant drug-related serious adverse events and long-term outcomes were favourable compared with historic cases. Three patients developed rebound hypertension on withdrawal of bosentan and one appeared to further benefit from maintenance therapy.
Upregulation of ET-1 ligand axis suggests that ET-1 receptor blockade is logical and treatment with bosentan appears to be safe in SRC. Future studies to assess therapeutic benefit and compare selective or non-selective receptor antagonists are justified.
Although macrophages are armed with potent antibacterial functions, Mycobacterium tuberculosis (Mtb) replicates inside these innate immune cells. Determinants of macrophage intrinsic bacterial ...control, and the Mtb strategies to overcome them, are poorly understood. To further study these processes, we used an affinity tag purification mass spectrometry (AP-MS) approach to identify 187 Mtb-human protein-protein interactions (PPIs) involving 34 secreted Mtb proteins. This interaction map revealed two factors involved in Mtb pathogenesis—the secreted Mtb protein, LpqN, and its binding partner, the human ubiquitin ligase CBL. We discovered that an lpqN Mtb mutant is attenuated in macrophages, but growth is restored when CBL is removed. Conversely, Cbl−/− macrophages are resistant to viral infection, indicating that CBL regulates cell-intrinsic polarization between antibacterial and antiviral immunity. Collectively, these findings illustrate the utility of this Mtb-human PPI map for developing a deeper understanding of the intricate interactions between Mtb and its host.
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•Creation of a Mtb-host protein-protein interaction map using mass spectrometry•LpqN is a novel Mtb virulence factor that associates with CBL, a host ubiquitin ligase•Removal of CBL rescues the attenuated lpqN Mtb mutant•CBL acts as a switch between antiviral and antibacterial responses in host
Penn et al. used an affinity tag purification mass spectrometry approach to generate an Mtb-human protein-protein interaction map, uncovering a connection between LpqN, a virulence factor in Mtb, and CBL, a host ubiquitin ligase. CBL suppresses lpqN attenuation and acts as a switch for host antibacterial and antiviral responses.
Background: Scleroderma renal crisis (SRC) is an important complication of systemic sclerosis, causing acute renal failure, and usually hypertension. Aims: To review the clinical and pathological ...features of SRC, and correlate them with renal outcomes and mortality. Design: Retrospective case series. Methods: We identified 110 cases of SRC managed at a single centre between 1990 and 2005. Results: SRC occurred in 5% of scleroderma cases under follow-up. Cases were predominantly female (81%), with diffuse cutaneous disease (78%). RNA polymerase antibodies were found in 59% of cases tested. Almost all (108/110) received treatment with ACE inhibitors (ACEIs). Dialysis was not required in 36%, was required temporarily (for up to 3 years) in 23%, was required permanently in 41%. Patients not on dialysis showed improvement in estimated glomerular filtration rate after SRC (mean change +23 ml/min over 3 years). Poor renal outcome was associated with lower blood pressure at presentation, and with higher age in those requiring dialysis. Steroid use, microangiopathic haemolytic anaemia, and antibody profile were not related to renal outcome. In the 58 renal biopsies available for clinical correlation, acute changes of mucoid intimal thickening in arteries and fibrinoid necrosis in arterioles were associated with a poorer renal outcome. Mortality was high (59% survival at 5 years), and was higher in men. Discussion: Despite the efficacy of ACEIs in managing SRC, the poor long-term outcome warrants evaluation for additional treatments for this devastating complication of systemic sclerosis.
Skeletal muscle differentiation is initiated by the transcription factor MyoD, which binds directly to the regulatory regions of genes expressed during skeletal muscle differentiation and initiates ...chromatin remodeling at specific promoters. It is not known, however, how MyoD initially recognizes its binding site in a chromatin context. Here we show that the H/C and helix III domains, two domains of MyoD that are necessary for the initiation of chromatin remodeling at the myogenin locus, together regulate a restricted subset of genes, including myogenin. These domains are necessary for the stable binding of MyoD to the myogenin promoter through an interaction with an adjacent protein complex containing the homeodomain protein Pbx, which appears to be constitutively bound at this site. This demonstrates a specific mechanism of targeting MyoD to loci in inactive chromatin and reveals a critical role of homeodomain proteins in marking specific genes for activation in the muscle lineage.
is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed ...a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the
effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in
-infected Lewis rat macrophages, consistent with the "NLRP1 functional degradation model." However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected
fitness in IFNγ-activated human fibroblasts, likely due to ITCH's role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses.IMPORTANCEEffector-triggered immunity represents an innate immune defense mechanism that plays a crucial role in sensing and controlling intracellular pathogen infection. The NLRP1 inflammasome in the Lewis rats can detect
infection, which triggers proptosis in infected macrophages and eliminates the parasite's replication niche. The work reported here revealed that host E3 ubiquitin ligase ITCH is able to recognize and interact with
effector protein GRA35 localized on the parasite-host interface, leading to NLRP1 inflammasome activation in Lewis rat macrophages. Furthermore, ITCH-GRA35 interaction contributes to the restriction of
in human fibroblasts stimulated by IFNγ. Thus, this research provides valuable insights into understanding pathogen recognition and restriction mediated by host E3 ubiquitin ligase.
We used expression arrays and chromatin immunoprecipitation assays to demonstrate that myogenesis consists of discrete subprograms of gene expression regulated by MyoD. Approximately 5% of assayed ...genes alter expression in a specific temporal sequence, and more than 1% are regulated by MyoD without the synthesis of additional transcription factors. MyoD regulates genes expressed at different times during myogenesis, and promoter-specific regulation of MyoD binding is a major mechanism of patterning gene expression. In addition, p38 kinase activity is necessary for the expression of a restricted subset of genes regulated by MyoD, but not for MyoD binding. The identification of distinct molecular mechanisms that regulate discrete subprograms of myogenesis should facilitate analyses of differentiation in normal development and disease.
This study followed the time-related changes in intestinal digestive physiology of post-smolt Atlantic salmon (Salmo salar L.; initial weight 500–600g) during the development of soybean meal induced ...enteritis. Intestinal tissue and content (chyme) were sampled after 1, 2, 3, 5, 7, 10, 14, 17, and 21days of dietary exposure to a 20% extracted soybean meal-containing (SBM) diet. Fish fed a fishmeal-based control diet sampled on day 0 (prior to SBM feeding) served as controls. From the different intestinal regions, histomorphological evaluation was conducted, activities of the pancreatic enzymes trypsin, chymotrypsin, elastase and lipase, as well as leucine aminopeptidase (LAP) were determined in chyme, and the brush border membrane (BBM) enzymes LAP and maltase were measured in homogenates of the intestinal wall. Epithelial cell functional differentiation was visually assessed using immunohistochemical expression of carbonic anhydrase 12 (CA-12). Histological signs of SBM-induced inflammation were observed in the distal intestine (DI) from day 5. At each given sampling time, pancreatic enzyme activities generally decreased aborally along the intestinal tract. Within each intestinal region, pancreatic protease activities tended to increase with exposure time to the SBM diet, with the greatest activity increases observed in the DI. The magnitude of the decrease in activity between the most proximal region and the most distal decreased with SBM exposure. DI tissue weight and BBM maltase and LAP activities were significantly reduced from day 1–2 and continued to decrease progressively with increasing SBM exposure time. Immunohistochemical staining of CA-12 also diminished over time starting at day 5. By day 7, very little CA-12 staining was observed. The data suggests an immediate and generalized physiological response to a change in diet and/or feed intake in all regions of the intestine, as well as a more localized functional loss in the DI that progressed with the development of SBM-induced inflammation from day 5. The diet change appeared to cause a rapid pancreatic response with increased secretion of all the enzymes investigated, while the longer term responses of the pancreatic proteases differed from that of lipase.
•Introducing SBM to the salmon diet caused changes in digestive processes.•The change in diet formulation and/or feed intake immediately affected intestines.•Pancreatic protease activities generally increased over time.•Pancreatic lipase and tissue enzyme activities remained unchanged or decreased.•Distal intestinal tissue responses reflected progression of inflammatory changes.
Macrophages employ an array of pattern recognition receptors to detect and eliminate intracellular pathogens that access the cytosol. The cytosolic carbohydrate sensors Galectin-3, -8, and -9 (Gal-3, ...Gal-8, and Gal-9) recognize damaged pathogen-containing phagosomes, and Gal-3 and Gal-8 are reported to restrict bacterial growth via autophagy in cultured cells. However, the contribution of these galectins to host resistance during bacterial infection in vivo remains unclear. We found that Gal-9 binds directly to Mycobacterium tuberculosis (Mtb) and Salmonella enterica serovar Typhimurium (Stm) and localizes to Mtb in macrophages. To determine the combined contribution of membrane damage-sensing galectins to immunity, we generated Gal-3, -8, and -9 triple knockout (TKO) mice. Mtb infection of primary macrophages from TKO mice resulted in defective autophagic flux but normal bacterial replication. Surprisingly, these mice had no discernable defect in resistance to acute infection with Mtb, Stm or Listeria monocytogenes, and had only modest impairments in bacterial growth restriction and CD4 T cell activation during chronic Mtb infection. Collectively, these findings indicate that while Gal-3, -8, and -9 respond to an array of intracellular pathogens, together these membrane damage-sensing galectins play a limited role in host resistance to bacterial infection.
An expansion of a CTG repeat at the DM1 locus causes myotonic dystrophy (DM) by altering the expression of the two adjacent genes, DMPK and SIX5, and through a toxic effect of the repeat-containing ...RNA. Here we identify two CTCF-binding sites that flank the CTG repeat and form an insulator element between DMPK and SIX5. Methylation of these sites prevents binding of CTCF, indicating that the DM1 locus methylation in congenital DM would disrupt insulator function. Furthermore, CTCF-binding sites are associated with CTG/CAG repeats at several other loci. We suggest a general role for CTG/CAG repeats as components of insulator elements at multiple sites in the human genome.
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