Inherited C-terminal TREX1 variants disrupt homology-directed repair to cause senescence and DNA damage phenotypes in Drosophila, mice, and humans.

Age-related microangiopathy, also known as small vessel disease (SVD), causes damage to the brain, retina, liver, and kidney. Based on the DNA damage theory of aging, we reasoned that genomic instability may underlie an SVD caused by dominant C-terminal variants in TREX1, the most abundant 3'-5' DNA exonuclease in mammals. C-terminal TREX1 variants cause an adult-onset SVD known as retinal vasculopathy with cerebral leukoencephalopathy (RVCL or RVCL-S). In RVCL, an aberrant, C-terminally truncated TREX1 mislocalizes to the nucleus due to deletion of its ER-anchoring domain. Since RVCL pathology mimics that of radiation injury, we reasoned that nuclear TREX1 would cause DNA damage. Here, we show that RVCL-associated TREX1 variants trigger DNA damage in humans, mice, and Drosophila, and that cells expressing RVCL mutant TREX1 are more vulnerable to DNA damage induced by chemotherapy and cytokines that up-regulate TREX1, leading to depletion of TREX1-high cells in RVCL mice. RVCL-associated TREX1 mutants inhibit homology-directed repair (HDR), causing DNA deletions and vulnerablility to PARP inhibitors. In women with RVCL, we observe early-onset breast cancer, similar to patients with BRCA1/2 variants. Our results provide a mechanistic basis linking aberrant TREX1 activity to the DNA damage theory of aging, premature senescence, and microvascular disease.

Regulation of cGAS and STING signaling during inflammation and infection.

Stimulator of interferon genes (STING) is a sensor of cyclic dinucleotides including cyclic GMP-AMP, which is produced by cyclic GMP-AMP synthase (cGAS) in response to cytosolic DNA. The cGAS-STING signaling pathway regulates both innate and adaptive immune responses, as well as fundamental cellular functions such as autophagy, senescence, and apoptosis. Mutations leading to constitutive activation of STING cause devastating human diseases. Thus, the cGAS-STING pathway is of great interest because of its role in diverse cellular processes and because of the potential therapeutic implications of targeting cGAS and STING. Here, we review molecular and cellular mechanisms of STING signaling, and we propose a framework for understanding the immunological and other cellular functions of STING in the context of disease.

The IFN-γ receptor promotes immune dysregulation and disease in STING gain-of-function mice.

STING gain-of-function mutations cause STING-associated vasculopathy with onset in infancy (SAVI) in humans, a disease characterized by spontaneous lung inflammation and fibrosis. Mice with STING gain-of-function mutations (SAVI mice) develop αß T cell-dependent lung disease and also lack lymph nodes. Although SAVI has been regarded as a type I interferonopathy, the relative contributions of the three interferon receptors are incompletely understood. Here, we show that STING gain of function led to upregulation of IFN-γ-induced chemokines in the lungs of SAVI mice and that deletion of the type II IFN receptor (IFNGR1), but not the type I IFN receptor (IFNAR1) or type III IFN receptor (IFNλR1), ameliorated lung disease and restored lymph node development in SAVI mice. Furthermore, deletion of IFNGR1, but not IFNAR1 or IFNλR1, corrected the ratio of effector to Tregs in SAVI mice and in mixed bone marrow chimeric mice. Finally, cultured SAVI mouse macrophages were hyperresponsive to IFN-γ, but not IFN-ß, in terms of Cxcl9 upregulation and cell activation. These results demonstrate that IFNGR1 plays a major role in autoinflammation and immune dysregulation mediated by STING gain of function.

Transferrable protection by gut microbes against STING-associated lung disease.

STING modulates immunity by responding to bacterial and endogenous cyclic dinucleotides (CDNs). Humans and mice with STING gain-of-function mutations develop a syndrome known as STING-associated vasculopathy with onset in infancy (SAVI), which is characterized by inflammatory or fibrosing lung disease. We hypothesized that hyperresponsiveness of gain-of-function STING to bacterial CDNs might explain autoinflammatory lung disease in SAVI mice. We report that depletion of gut microbes with oral antibiotics (vancomycin, neomycin, and ampicillin [VNA]) nearly eliminates lung disease in SAVI mice, implying that gut microbes might promote STING-associated autoinflammation. However, we show that germ-free SAVI mice still develop severe autoinflammatory disease and that transferring gut microbiota from antibiotics-treated mice to germ-free animals eliminates lung inflammation. Depletion of anaerobes with metronidazole abolishes the protective effect of the VNA antibiotics cocktail, and recolonization with the metronidazole-sensitive anaerobe Bacteroides thetaiotaomicron prevents disease, confirming a protective role of a metronidazole-sensitive microbe in a model of SAVI.

STING-associated lung disease in mice relies on T cells but not type I interferon.

BACKGROUND: Monogenic interferonopathies are thought to be mediated by type I interferon. For example, a gain-of-function mutation in stimulator of interferon genes (STING; N153S) upregulates type I interferon-stimulated genes and causes perivascular inflammatory lung disease in mice. The equivalent mutation in human subjects also causes lung disease, which is thought to require signaling through the cyclic GMP-AMP synthase (cGAS)-STING pathway and subsequent activation of interferon regulatory factors (IRFs) 3 and 7, type I interferon, and interferon-stimulated genes. OBJECTIVE: We set out to define the roles of cGAS, IRF3, IRF7, the type I interferon receptor (IFN-α and IFN-ß receptor subunit 1 [IFNAR1]), T cells, and B cells in spontaneous lung disease in STING N153S mice. METHODS: STING N153S mice were crossed to animals lacking cGAS, IRF3/IRF7, IFNAR1, adaptive immunity, αß T cells, and mature B cells. Mice were evaluated for spontaneous lung disease. Additionally, bone marrow chimeric mice were assessed for lung disease severity and survival. RESULTS: Lung disease in STING N153S mice developed independently of cGAS, IRF3/IRF7, and IFNAR1. Bone marrow transplantation revealed that certain features of STING N153S-associated disease are intrinsic to the hematopoietic compartment. Recombination-activating gene 1 (Rag1)-/- STING N153S mice that lack adaptive immunity had no lung disease, and T-cell receptor ß chain (Tcrb)-/- STING N153S animals only had mild disease. STING N153S led to a reduction in percentages and numbers of naive and regulatory T cells, as well as an increased frequency of cytokine-producing effector T cells. CONCLUSION: Spontaneous lung disease in STING N153S mice develops independently of type I interferon signaling and cGAS. STING N153S relies primarily on T cells to promote lung disease in mice.

A unique role for galectin-9 in angiogenesis and inflammatory arthritis.

BACKGROUND: Galectin-9 (Gal-9) is a mammalian lectin secreted by endothelial cells that is highly expressed in rheumatoid arthritis synovial tissues and synovial fluid. Roles have been proposed for galectins in the regulation of inflammation and angiogenesis. Therefore, we examined the contribution of Gal-9 to angiogenesis and inflammation in arthritis. METHODS: To determine the role of Gal-9 in angiogenesis, we performed human dermal microvascular endothelial cell (HMVEC) chemotaxis, Matrigel tube formation, and mouse Matrigel plug angiogenesis assays. We also examined the role of signaling molecules in Gal-9-induced angiogenesis by using signaling inhibitors and small interfering RNA (siRNA). We performed monocyte (MN) migration assays in a modified Boyden chamber and assessed the arthritogenicity of Gal-9 by injecting Gal-9 into mouse knees. RESULTS: Gal-9 significantly increased HMVEC migration, which was decreased by inhibitors of extracellular signal-regulating kinases 1/2 (Erk1/2), p38, Janus kinase (Jnk), and phosphatidylinositol 3-kinase. Gal-9 HMVEC-induced tube formation was reduced by Erk1/2, p38, and Jnk inhibitors, and this was confirmed by siRNA knockdown. In mouse Matrigel plug assays, plugs containing Gal-9 induced significantly higher angiogenesis, which was attenuated by a Jnk inhibitor. Gal-9 also induced MN migration, and there was a marked increase in MN ingress when C57BL/6 mouse knees were injected with Gal-9 compared with the control, pointing to a proinflammatory role for Gal-9. CONCLUSIONS: Gal-9 mediates angiogenesis, increases MN migration in vitro, and induces acute inflammatory arthritis in mice, suggesting a novel role for Gal-9 in angiogenesis, joint inflammation, and possibly other inflammatory diseases.

A Truncated IL-17RC Peptide Ameliorates Synovitis and Bone Destruction of Arthritic Mice.

Peptide-based therapy, such as modified peptides, has attracted increased attention. IL-17 is a promising therapeutic target for autoimmune diseases, and levels of circulating bioactive IL-17 are associated with rheumatoid arthritis severity. In this study, a modified truncated IL-17RC is generated to ameliorate inflammation and bone destruction in arthritis. The truncated IL-17RC binds to both IL-17A and IL-17F with higher binding capacity compared to nonmodified IL-17RC. In addition, the truncated IL-17RC reduces the secretion of inflammatory and osteoclastogenic factors induced by IL-17A/F in vitro. Moreover, the administration of truncated IL-17RC dramatically improves symptoms of inflammation and inhibited bone destruction in collagen-induced arthritis mice. Collectively, these data demonstrate that modified truncated IL-17RC peptide may be a more effective treatment strategy in the simultaneous inhibition of both IL-17A and IL-17F signaling, whereas the existing agents neutralize IL-17A or IL-17F alone. These suggest that the truncated IL-17RC may be a potential candidate in the treatment of inflammatory associated bone diseases.

Inflammatory properties of inhibitor of DNA binding 1 secreted by synovial fibroblasts in rheumatoid arthritis.

BACKGROUND: Inhibitor of DNA binding 1 (Id1) is a nuclear protein containing a basic helix-loop-helix (bHLH) domain that regulates cell growth by selective binding and prevention of gene transcription. Sources of Id1 production in rheumatoid arthritis synovial tissue (RA ST) and its range of functional effects in RA remain to be clarified. METHODS: We analyzed Id1 produced from synovial fibroblasts and endothelial cells (ECs) with histology and real-time polymerase chain reaction (RT-PCR). Fibroblast supernatants subjected to differential centrifugation to isolate and purify exosomes were measured for Id1 by enzyme-linked immunosorbent assay (ELISA). Western blotting of Id1-stimulated ECs was performed to determine the kinetics of intracellular protein phosphorylation. EC intracellular signaling pathways induced by Id1 were subsequently targeted with silencing RNA (siRNA) for angiogenesis inhibition. RESULTS: By PCR and histologic analysis, we found that the primary source of Id1 in STs is from activated fibroblasts that correlate with inflammatory scores in human RA ST and in joints from K/BxN serum-induced mice. Normal (NL) and RA synovial fibroblasts increase Id1 production with stimulation by transforming growth factor beta (TGF-ß). Most of the Id1 released by RA synovial fibroblasts is contained within exosomes. Endothelial progenitor cells (EPCs) and human dermal microvascular ECs (HMVECs) activate the Jnk signaling pathway in response to Id1, and Jnk siRNA reverses Id1-induced HMVEC vessel formation in Matrigel plugs in vivo. CONCLUSIONS: Id1 is a pleotropic molecule affecting angiogenesis, vasculogenesis, and fibrosis. Our data shows that Id1 is not only an important nuclear protein, but also can be released from fibroblasts via exosomes. The ability of extracellular Id1 to activate signaling pathways expands the role of Id1 in the orchestration of tissue inflammation.

A key role for Fut1-regulated angiogenesis and ICAM-1 expression in K/BxN arthritis.

OBJECTIVES: Angiogenesis contributes to the pathogenesis of rheumatoid arthritis. Fucosyltransferases (Futs) are involved in angiogenesis and tumour growth. Here, we examined the role of Fut1 in angiogenesis and K/BxN serum transfer arthritis. METHODS: We examined Fut1 expression in human dermal microvascular endothelial cells (HMVECs) by quantitative PCR. We performed a number of angiogenesis assays to determine the role of Fut1 using HMVECs, Fut1 null (Fut1(-/-)), and wild type (wt) endothelial cells (ECs) and mice. K/BxN serum transfer arthritis was performed to determine the contribution of Fut1-mediated angiogenesis in Fut1(-/-) and wt mice. A static adhesion assay was implemented with RAW264.7 (mouse macrophage cell line) and mouse ECs. Quantitative PCR, immunofluorescence and flow cytometry were performed with Fut1(-/-) and wt ECs for adhesion molecule expression. RESULTS: Tumour necrosis factor-α induced Fut1 mRNA and protein expression in HMVECs. HMVECs transfected with Fut1 antisense oligodeoxynucleotide and Fut1(-/-) ECs formed significantly fewer tubes on Matrigel. Fut1(-/-) mice had reduced angiogenesis in Matrigel plug and sponge granuloma angiogenesis assays compared with wt mice. Fut1(-/-) mice were resistant to K/BxN serum transfer arthritis and had decreased angiogenesis and leucocyte ingress into inflamed joints. Adhesion of RAW264.7 cells to wt mouse ECs was significantly reduced when Fut1 was lacking. Fut1(-/-) ECs had decreased intercellular adhesion molecule-1 (ICAM-1) expression at mRNA and protein levels compared with wt ECs. ICAM-1 was also decreased in Fut1(-/-) arthritic ankle cryosections compared with wt ankles. CONCLUSIONS: Fut1 plays an important role in regulating angiogenesis and ICAM-1 expression in inflammatory arthritis.

Fucosyltransferase 1 mediates angiogenesis in rheumatoid arthritis.

OBJECTIVE: To determine the role of α(1,2)-linked fucosylation of proteins by fucosyltransferase 1 (FUT1) in rheumatoid arthritis (RA) angiogenesis. METHODS: Analysis of α(1,2)-linked fucosylated proteins in synovial tissue (ST) samples was performed by immunohistologic staining. Expression of α(1,2)-linked fucosylated angiogenic chemokine in synovial fluid (SF) was determined by immunoprecipitation and lectin blotting. To determine the angiogenic role of α(1,2)-linked fucosylated proteins in RA, we performed human dermal microvascular endothelial cell (HMVEC) chemotaxis and Matrigel assays using sham-depleted and α(1,2)-linked fucosylated protein-depleted RA SF samples. To examine the production of proangiogenic chemokines by FUT1 in HMVECs, cells were transfected with FUT1 sense or antisense oligonucleotides, and enzyme-linked immunosorbent assay was performed. We then studied mouse lung endothelial cell (EC) chemotaxis using wild-type and FUT1 gene-deficient mouse lung ECs. RESULTS: RA ST endothelial cells showed high expression of α(1,2)-linked fucosylated proteins compared to normal ST. The expression of α(1,2)-linked fucosylated monocyte chemoattractant protein 1 (MCP-1)/CCL2 was significantly elevated in RA SF compared with osteoarthritis SF. Depletion of α(1,2)-linked fucosylated proteins in RA SF induced less HMVEC migration and tube formation than occurred in sham-depleted RA SF. We found that blocking FUT1 expression in ECs resulted in decreased MCP-1/CCL2 and RANTES/CCL5 production. Finally, we showed that FUT1 regulates EC migration in response to vascular endothelial cell growth factor. CONCLUSION: Our findings indicate that α(1,2)-linked fucosylation by FUT1 may be an important new target for angiogenic diseases such as RA.

Pagetoid variant of actinic keratosis with or without squamous cell carcinoma of sun-exposed skin: a lesion simulating extramammary Paget's disease.

BACKGROUND: Extramammary Paget's disease usually occurs in anogenital skin. We present five cases of squamous cell carcinoma in situ of sun-exposed skin and non-squamous cell carcinoma in situ actinic keratosis that displayed atypical keratinocytes disposed in intraepithelial cell nests and immunohistochemical staining simulating extramammary Paget's disease. METHODS AND RESULTS: Two pilot cases--one squamous cell carcinoma in situ and one non-squamous cell carcinoma in situ actinic keratosis with formation of intra-epidermal nests of atypical keratinocytes with a pagetoid spread pattern--were encountered at our institution. Fifty-four consecutive cases of squamous cell carcinoma in situ including bowenoid actinic keratosis and 34 cases of non-squamous cell carcinoma in situ actinic keratosis were reviewed to identify pagetoid spread of atypical cells. Representative sections of all cases with pagetoid spread of atypical keratinocytes were submitted for special stains for mucin, and immunostaining for cytokeratin 7 (CK7), cytokeratin 20 (CK20), cytokeratin CAM 5.2 (CAM 5.2), carcinoembryonic antigen (CEA), vimentin and S100 protein. In the group of squamous cell carcinoma in situ, 10 cases displayed pagetoid spread of atypical keratinocytes with cytoplasm ranging from clear to pale and atypical hyperchromatic nuclei. One review squamous cell carcinoma in situ was multicentric with three separate lesions. The atypical keratinocytes tended to form well to poorly defined cell groups extending from the basal cell layer to the corneal layer. No similar cases were identified in the group of non-squamous cell carcinoma in situ actinic keratosis. Two pilot cases and three of 10 review cases with a total of seven separate lesions displayed a moderate to marked immunohistochemical reactivity for CK7 similar to extramammary Paget's disease. CEA immunoreactivity was also detected in two of these cases. In addition, two of 44 squamous cell carcinomas in situ without pagetoid spread of atypical keratinocytes showed a moderate reactivity for CK7 in very occasional atypical keratinocytes. The remaining seven squamous cell carcinomas in situ with pagetoid spread of atypical keratinocytes were not immunoreactive for CEA and CK7. Immunostaining for CK20, vimentin, S100 protein was negative in all atypical cells in all study cases. CONCLUSIONS: Actinic keratosis, particularly squamous cell carcinoma in situ of sun-exposed skin, may have histopathological and immunohistochemical features similar to extramammary Paget's disease and probably represents a variant of actinic keratosis. Awareness of the pagetoid variant of actinic keratosis arising in sun-exposed skin is helpful to avoid the over-diagnosis of extramammary Paget's disease.

Aortic medial changes associated with bicuspid aortic valve: myth or reality?

OBJECTIVE: To determine whether aortic medial changes are more severe in patients who require aortic valve replacement of congenitally bicuspid aortic valves (BAV) than in patients who require replacement of tricuspid aortic valves and acquired valvular disease (AVD). DESIGN: Aortas from autopsies of 14 patients with BAV and 25 with AVD were histologically assessed by two 'blinded' cardiovascular pathologists and analyzed independently with computer-aided morphometry. The aortic valves were examined for valvular fibrosis and calcification. SETTING: The patient population was from a tertiary-care facility. PATIENTS: Patients were selected by retrospective review of autopsy records for patient deaths after aortic valve replacement, over the period 1984 to 1995. RESULTS: There were no significant differences in age (P=0.89), sex (P=0.94), prevalence of systemic arterial hypertension (P=0.37), valvular degenerative changes (P=0.10 and P=1.0) or heart weights (P=0.60) between the two groups. Histological scores for aortic medial degenerative changes including elastic fragmentation, fibrosis and medionecrosis were not statistically different between the groups. However, morphometry demonstrated less elastic tissue in patients with BAV (P=0.003). CONCLUSION: Routine microscopy shows no significant difference in the degree of aortic medial degenerative changes between patients with BAV and AVD. However, morphometry shows less elastic tissue in the aortas of BAV patients. This may explain the anecdotal increase in aortic fragility and propensity for aortic dissection in these patients.

Reliability of transthoracic echocardiography in the assessment of aortic valve morphology: pathological correlation in 178 patients.

OBJECTIVE: To examine patient characteristics affecting the utility of transthoracic echocardiography in determining aortic valve morphology, particularly for the diagnosis of congenital bicuspid aortic valve (BAV). DESIGN: A retrospective comparison of preoperative echocardiographic determination of aortic valve morphology with pathological findings of the explanted valves. SETTING: A tertiary referral centre. PATIENTS: Consecutive patients who had aortic valve replacement between July 1994 and April 1996, and had preoperative echocardiograms. RESULTS: Of 313 patients, 181 (58%) had preoperative echocardiography. Three of the valves were excluded because they were too fragmented for pathological determination of valvular morphology. In the remaining 178 patients, aortic valvular morphology was determined by echocardiography in 104 (58%). Multivariate analysis showed that echocardiography was successful less often in women (odds ratio 0.44, P = 0.03) and in patients with densely calcified valves (odds ratio 0.69, P = 0.02), whereas age had no effect (odd ratio 0.99, P = 0.42). In those with adequate echocardiographic images, echocardiography had both a high sensitivity (0.92) and a high specificity (0.96) for the diagnosis of BAV. CONCLUSIONS: Echocardiography is a useful tool for the diagnosis of BAV, although suboptimal images may pose a problem in many patients, particularly women and patients with heavily calcified valves. When adequate images are obtained, transthoracic echocardiography can reliably identify aortic valvular morphology in most patients.

Implantable cardioverter-defibrillators and the pathologist: comment and cautionary notes.

This paper briefly reviews the components of, the clinical uses of, the techniques to place, and the complications related to implantable cardioverter-defibrillators (ICDs). Information useful in the specific identification of ICDs is presented. A series of recommendations for the autopsy examination or postmortem explantation of ICDs by the pathologist is given. Because of the serious risk of injury to the pathologist possible with postmortem discharges of ICDs which have not been deactivated, and because of the risk of device explosion if the ICD is incinerated, a number of cautionary notes are provided. A brief case with occurrence of accidental postmortem discharge of an active ICD is also presented.

Assessing the quality of the supervision of reproductive health services in Uganda.

PIP: The Quality Assurance Unit (QAU) of Uganda's Ministry of Health (MOH) has promoted quality of care in Uganda for a number of years. The US Agency for International Development (USAID) has also had a major role in promoting quality of care in family planning, the case management of sexually transmitted diseases, and maternal care, working mainly through reproductive health projects implemented by the nongovernmental organizations (NGOs) Pathfinder, AMREF, and Care. These projects have all emphasized training as the main way to build worker competence and have promoted supervision as a means for quality control and improvement. As part of an overall review of reproductive health project activities, USAID asked the Quality Assurance Project (QAP) to help review supervisory effectiveness and data quality. QAP therefore collaborated with the MOH QAU to design and conduct a quality assessment of reproductive health supervision, examining the use of supervision tools, the frequency and quality of supervisory encounters, and the use of supervision data and sustainability. The assessment found that each of the three NGO projects has made significant achievements in developing a strategy for the quality-focused supervision of reproductive health services. However, several areas were also found in which supervision could be enhanced, especially those related to supervision tools and the use of the data they generate, and the integration of NGO project and district health team supervision.^ieng

Quality supervision.

PIP: Although supervision is often identified as the vehicle through which the quality of health care services can be assured, it typically receives neither the human nor financial support needed to fully conduct and sustain the necessary supervisory activities. In the current decentralization of health services management occurring in many countries, full responsibility for the supervision of facility and community health workers has been shifted to area and district levels, often without providing the training and resources needed to undertake supervisory functions. Furthermore, the activities with which supervisors are charged are often poorly defined. Health care systems have a wide range of options in developing a locally appropriate and sustainable supervision strategy at the primary level. Key issues are who supervises and how often, and the use of supervisory job aids in measuring the quality of care. The quality assurance approach to supervision and the implementation of quality-focused supervision are discussed.^ieng