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Objective The coronavirus disease 2019 (COVID-19) pandemic created challenges for patients with RA. We examined the potential impact of the pandemic on patient-reported outcomes (PROs), disease activity and medication profiles, comparing the periods pre-pandemic and during the pandemic. Methods Patients enrolled in the Ontario Best Practices Research Initiative were included if they had at least one visit to a physician or study interviewer within 12 months before and after the start of pandemic-related closures in Ontario (15 March 2020). Baseline characteristics, disease activity, PROs [i.e. health assessment questionnaire disability index, RA disease activity index (RADAI), European quality of life five-dimension questionnaire], medication use and changes were included. Student’s paired two-sample t-tests and McNamar’s tests were performed for continuous and categorical variables between time periods. Results The sample for analysis consisted of 1508 patients, with a mean (s.d.) age of 62.7 (12.5) years, and 79% were female. Despite decreases in the number of in-person visits during the pandemic, there was no significant negative impact on disease activity or PRO scores. The DASs in both periods remained low, with either no clinically significant differences or slight improvement. Scores for mental, social and physical health were either stable or improved. There were statistically significant decreases in conventional synthetic DMARD use (P Conclusion In this cohort, disease activity and PROs of RA patients remained stable during the COVID-19 pandemic. The longer-term outcomes of the pandemic warrant investigation.

The tourism industry is in a fight for survival during the coronavirus pandemic. This essay was written based upon several interviews conducted with directors and general managers of nine well-known hotels in China with the aim to investigate what new technologies are used to mitigate the impact of the pandemic. DeLone and McLean’s Information System Success Model was applied to examine the adopted digital technologies. Live-stream promotion and live-stream conference are introduced to primarily improve information quality, while 5G technology and Wi-Fi 6 are installed to enhance the system quality. Facial recognition, AI, and Robots are integrated to the daily operations to enhance service quality. Challenges and future directions are discussed.

Medical cannabis is often used to alleviate common symptoms in patients with chronic conditions. With cannabis legalisation in Canada and easier access, it is important that rheumatologists understand its potential impact on their practice. Among patients attending rheumatology clinics in Ontario we assessed: the prevalence of medical cannabis use; symptoms treated; rheumatologists' perceptions.Eight rheumatology clinics recruited consecutive adult patients in a 3-part medical cannabis survey: the first completed by rheumatologists; the second by all patients; the third by medical cannabis users. Student's t-test and Chi-square test were used to compare medical cannabis users to never users.799 patients participated, 163 (20.4%) currently using medical cannabis or within2 years and 636 never users; most had rheumatoid arthritis (37.8%) or osteoarthritis (34.0%). Compared to never users, current/past-users were younger; more likely to be taking opioids/anti-depressants, have psychiatric/gastrointestinal disorders, and have used recreational cannabis (p0.05); had higher physician (2.9 vs. 2.1) and patient (6.0 vs. 4.2) global scores, and pain (6.2 vs. 4.7) (p0.0001). Pain (95.5%), sleeping (82.3%) and anxiety (58.9%) were the most commonly treated symptoms; 78.2% of current/past-users reported medical cannabis was at least somewhat effective. Most rheumatologists reported being uncomfortable to authorise medical cannabis, primarily due to lack of evidence, knowledge, and product standardisation.Medical cannabis use among rheumatology patients in Ontario was two-fold higher than that reported for the general population of similar age. Use was associated with more severe disease, pain, and prior recreational use. Reported lack of research, knowledge, and product standardisation were barriers for rheumatologist use authorisation.

The mechanisms that drive leukocyte recruitment to the kidney are incompletely understood. Dipeptidase-1 (DPEP1) is a major neutrophil adhesion receptor highly expressed on proximal tubular cells and peritubular capillaries of the kidney. Renal ischemia reperfusion injury (IRI) induces robust neutrophil and monocyte recruitment and causes acute kidney injury (AKI). Renal inflammation and the AKI phenotype were attenuated in Dpep1 −/− mice or mice pretreated with DPEP1 antagonists, including the LSALT peptide, a nonenzymatic DPEP1 inhibitor. DPEP1 deficiency or inhibition primarily blocked neutrophil adhesion to peritubular capillaries and reduced inflammatory monocyte recruitment to the kidney after IRI. CD44 but not ICAM-1 blockade also decreased neutrophil recruitment to the kidney during IRI and was additive to DPEP1 effects. DPEP1, CD44, and ICAM-1 all contributed to the recruitment of monocyte/macrophages to the kidney following IRI. These results identify DPEP1 as a major leukocyte adhesion receptor in the kidney and potential therapeutic target for AKI.

Absent in melanoma-2 (AIM2) is an inflammasome-forming innate immune sensor for dsDNA but also exhibits inflammasome-independent functions such as restricting cellular proliferation. AIM2 is expressed in the kidney, but its localization and function are not fully characterized. In normal human glomeruli, AIM2 localized to podocytes. In patients with glomerulonephritis, AIM2 expression increased in CD44+-activated parietal epithelial cells within glomerular crescents. To explore AIM2 effects in glomerular disease, studies in Aim2−/− mice were performed. Aim2−/− glomeruli showed reduced expression of Wilm tumor gene-1 (WT1), WT1-driven podocyte genes, and increased proliferation in outgrowth assays. In a nephrotoxic serum (NTS)–induced glomerulonephritis model, Aim2−/− (B6) mice exhibited more severe glomerular crescent formation, tubular injury, inflammation, and proteinuria compared with wild-type controls. Inflammasome activation markers were absent in both Aim2−/− and wild-type kidneys, despite an increased inflammatory transcriptomic signature in Aim2−/− mice. Aim2−/− mice also demonstrated dysregulated cellular proliferation and an increase in CD44+ parietal epithelial cells during glomerulonephritis. The augmented inflammation and epithelial cell proliferation in Aim2−/− (B6) mice was not due to genetic background, as Aim2−/− (B6.129) mice demonstrated a similar phenotype during NTS glomerulonephritis. The AIM2-like receptor (ALR) locus was necessary for the inflammatory glomerulonephritis phenotype observed in Aim2−/− mice, as NTS-treated ALR−/− mice displayed equal levels of injury as wild-type controls. Podocyte outgrowth from ALR−/− glomeruli was still increased, however, confirming that the ALR locus is dispensable for AIM2 effects on epithelial cell proliferation. These results identify a noncanonical role for AIM2 in suppressing inflammation and epithelial cell proliferation during glomerulonephritis.

The NLRP6 innate immune sensor is regulated by tissue-selective alternate promoters that facilitate translational gene silencing outside of the intestinal epithelium in both humans and mice., The pryin domain (PYD) domain is involved in protein interactions that lead to assembly of immune-sensing complexes such as inflammasomes. The repertoire of PYD-containing genes expressed by a cell type arms tissues with responses against a range of stimuli. The transcriptional regulation of the PYD gene family however is incompletely understood. Alternative promoter utilization was identified as a mechanism regulating the tissue distribution of human PYD gene family members, including NLRP6 that is translationally silenced outside of intestinal tissue. Results show that alternative transcriptional promoters mediate NLRP6 silencing in mice and humans, despite no upstream genomic synteny. Human NLRP6 contains an internal alternative promoter within exon 2 of the PYD, resulting in a truncated mRNA in nonintestinal tissue. In mice, a proximal promoter was used that expanded the 5′ leader sequence restricting nuclear export and abolishing translational efficiency. Nlrp6 was dispensable in disease models targeting the kidney, which expresses noncanonical isoforms. Thus, alternative promoter use is a critical mechanism not just for isoform modulation but for determining expression profile and function of PYD family members.

BackgroundCOVID-19 can lead to acute respiratory failure and an exaggerated inflammatory response. Studies have suggested promising outcomes using monoclonal antibodies targeting IL-1β (Anakinra) or IL6 (Tocilizumab), however no head to head comparison was done between the two treatments. Herein, we report our experience in treating COVID-19 pneumonia associated with cytokine storm with either subcutaneous Anakinra given concomitantly with intravenous immunoglobulin (IVIG), or intravenous Tocilizumab.MethodsComprehensive clinical and laboratory data from patients with COVID-19 pneumonia admitted at our hospital between March and May 2020 were collected. Patients who received either Anakinra/ IVIG or Tocilizumab were selected. Baseline characteristics including oxygen therapy, respiratory status evaluation using ROX index, clinical assessment using NEWS score and laboratory data were collected. Outcomes included mortality, intubation, ICU admission and length of stay. In addition, we compared the change in ROX index, NEWS score and inflammatory markers at days 7 and 14 post initiation of therapy.Results84 consecutive patients who received either treatment (51 in the Anakinra/ IVIG group and 33 in the Tocilizumab group) were retrospectively studied. Baseline inflammatory markers were similar in both groups. There was no significant difference regarding to death (21.6% vs 15.2%, p 0.464), intubation (15.7% vs 24.2%, p 0.329), ICU need (57.1% vs 48.5%, p 0.475) or length of stay (13+9.6 vs 14.9+11.6, p 0.512) in the Anakinra/IVIG and Tocilizumab, respectively. Additionally, the rate of improvement in ROX index, NEWS score and inflammatory markers was similar in both groups at days 7 and 14. Furthermore, there was no difference in the incidence of superinfection in both groups.ConclusionTreating COVID-19 pneumonia associated with cytokine storm features with either subcutaneous Anakinra/IVIG or intravenous Tocilizumab is associated with improved clinical outcomes in most subjects. The choice of treatment does not appear to affect morbidity or mortality. Randomized controlled trials are needed to confirm our study findings.FundingNone.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition caused by overproduction of inflammatory cytokines and overactivation of macrophages that can progress to multiorgan dysfunction and failure. Although there are guidelines that attempt to recognize the condition in its early stage, diagnosis can be very challenging due to heterogeneous presentations of HLH. Symptoms and clinical findings include fever, neurologic complaints, respiratory issues, liver dysfunction, cytopenias, amongst others most of which are not specific to HLH. In addition, response to treatment can be highly variable, necessitating an individualized treatment plan based on the presentation. We present a case of a 21-year-old female with a history of biopsy-proven inflammatory myositis on azathioprine and prednisone who presented with fever, hypotension, and pancytopenia. Additional imaging studies showed multiorgan involvement, including pneumonia, pyelonephritis, and splenomegaly. A bone marrow biopsy of her iliac crest showed hemophagocytosis and the infectious workup confirmed cytomegalovirus (CMV) infection, which led to the diagnosis of CMV-induced HLH. She was treated initially with anakinra for macrophage activation syndrome (MAS) in addition to dexamethasone and ganciclovir. Unfortunately, she did not respond to anakinra and was subsequently switched to etoposide with dexamethasone and valganciclovir, which subsequently helped our patient to recover clinically. Our case highlights the challenging nature of HLH and the importance of early detection and a personalized treatment plan in achieving optimal outcomes in patients with HLH.

Central nervous system (CNS) vasculopathy caused by varicella zoster virus (VZV) is a rare condition. Rarer still is the development of CNS vasculopathy in the absence of a typical zoster rash, a phenomenon known as zoster sine herpete. We report a case of a 34-year-old male with HIV, non-compliant with highly active antiretroviral therapy (HAART), who presented with left-sided temporal headaches and numbness without rash. The patient had a complicated one-month hospital stay when he was initially diagnosed with mycobacterium avium complex (MAC) tuberculosis infection and treated with isoniazid, rifabutin, ethambutol, and azithromycin. Additionally, he was thought to have immune reconstitution inflammatory syndrome (IRIS) and was given steroids. Unfortunately, he presented one day post-discharge with lethargy, aphasia, and dysphagia and was found to have acute/subacute infarcts affecting multiple areas of the brain. CT angiogram (CTA) of the brain showed evidence of multifocal areas of mild to moderate stenosis throughout the intracranial arterial circulation. The patient underwent conventional angiography, which showed segmental arterial constrictions with post-stenotic dilatation consistent with vasculitis. Cerebrospinal fluid (CSF) studies eventually returned positive for VZV by polymerase chain reaction (PCR), confirming a diagnosis of VZV-induced CNS vasculopathy, or more specifically, CNS vasculopathy due to zoster sine herpete. The patient was treated with high-dose steroids as well as IV acyclovir with improvement in his symptoms. He was discharged with advice for a close follow-up with the infectious disease (ID) department. Our case highlights the importance of maintaining a high index of suspicion for varicella infection masquerading as CNS vasculitis, particularly in the absence of classic blistering shingles rash. Early detection may prevent neurological sequelae of the infection, including stroke, dissection, or neuropathy.

Summary: The non-canonical caspase-4 and canonical NLRP3 inflammasomes are both activated by intracellular lipopolysaccharide (LPS), but the crosstalk between these two pathways remains unclear. Shiga toxin 2 (Stx2)/LPS complex, from pathogenic enterohemorrhagic Escherichia coli, activates caspase-4, gasdermin D (GSDMD), and the NLRP3 inflammasome in human THP-1 macrophages, but not mouse macrophages that lack the Stx receptor CD77. Stx2/LPS-mediated IL-1β secretion and pyroptosis are dependent on mitochondrial reactive oxygen species (ROS) downstream of the non-canonical caspase-4 inflammasome and cleaved GSDMD, which is enriched at the mitochondria. Blockade of caspase-4 activation and ROS generation as well as GSDMD deficiency significantly reduces Stx2/LPS-induced IL-1β production and pyroptosis. The NLRP3 inflammasome plays a significant role in amplifying Stx2/LPS-induced GSDMD cleavage and pyroptosis, with significant reduction of these responses in NLRP3-deficient THP-1 cells. Together, these data show that Stx2/LPS complex activates the non-canonical inflammasome and mitochondrial ROS upstream of the NLRP3 inflammasome to promote cytokine maturation and pyroptosis. : Shiga toxin 2 is a major virulence factor of enterohemorrhagic E. coli. Platnich et al. show that Shiga toxin 2 and co-transported lipopolysaccharide activate caspase-4, gasdermin D, and mitochondrial ROS upstream of the NLRP3 inflammasome in macrophages. Inflammasome activation may play a role in the pathogenesis of enterohemorrhagic E. coli infection and disease. Keywords: Shiga toxin, inflammasome, NLRP3, caspase-4, gasdermin D, enterohemorrhagic Escherichia coli, macrophages

Oncolytic virus (OV) therapy is an emerging cancer treatment that uses replicating viruses to infect and kill tumor cells and incite anticancer immunity. While the approach shows promise, it currently fails most patients, indicating strategies to improve OV activity are needed. Developing these will require greater understanding of OV biology, particularly in the context of OV delivery and clearance, the infection process within a complex tumor microenvironment, and the modulation of anticancer immunity. To help achieve this, we have established a technique for high-resolution 4D imaging of OV-host interactions within intact tissues of live mice using intravital microscopy (IVM). We show that oncolytic vesicular stomatitis virus (VSV) directly labeled with Alexa Fluor dyes is easily visualized by single- or multiphoton microscopy while retaining bioactivity in vivo. The addition of fluorophore-tagged antibodies and genetically encoded reporter proteins to image target cells and the virus infection enables real-time imaging of dynamic interactions between VSV and host cells in blood, tumor, and visceral organs of live mice. The method has sufficient in vivo resolution to observe leukocytes in blood binding to and transporting VSV particles, foci of VSV infection spreading through a tumor, and antigen-presenting cells in the spleen interacting with and being infected by VSV. Visualizing OV-host interactions by IVM represents a powerful new tool for studying OV therapy. Keywords: oncolytic virus, intravital microscopy, virus-host interactions, tumor microenvironment, secondary lymphoid organs

Inflammation is an integral component of many diseases, including chronic kidney disease (CKD). ASC (apoptosis-associated speck-like protein containing CARD, also PYCARD) is the key inflammasome adaptor protein in the innate immune response. Since ASC specks, a macromolecular condensate of ASC protein, can be released by inflammasome-activated cells into the extracellular space to amplify inflammatory responses, the ASC protein could be an important biomarker in diagnostic applications. Herein, we describe the development and validation of a multiple reaction monitoring mass spectrometry (MRM-MS) assay for the accurate quantification of ASC in human biospecimens. Limits of detection and quantification for the signature DLLLQALR peptide (used as surrogate for the target ASC protein) were determined by the method of standard addition using synthetic isotope-labeled internal standard (SIS) peptide and urine matrix from a healthy donor (LOQ was 8.25 pM, with a ~ 1000-fold linear range). We further quantified ASC in the urine of CKD patients (8.4 ± 1.3 ng ASC/ml urine, n = 13). ASC was positively correlated with proteinuria and urinary IL-18 in CKD samples but not with urinary creatinine. Unfortunately, the ASC protein is susceptible to degradation, and patient urine that was thawed and refrozen lost 85% of the ASC signal. In summary, the MRM-MS assay provides a robust means to quantify ASC in biological samples, including clinical biospecimens; however, sample collection and storage conditions will have a critical impact on assay reliability.

Nonmicrobial inflammation contributes to CKD progression and fibrosis. Absent in melanoma 2 (AIM2) is an inflammasome-forming receptor for double-stranded DNA. AIM2 is expressed in the kidney and activated mainly by macrophages. We investigated the potential pathogenic role of the AIM2 inflammasome in kidney disease. In kidneys from patients with diabetic or nondiabetic CKD, immunofluorescence showed AIM2 expression in glomeruli, tubules, and infiltrating leukocytes. In a mouse model of unilateral ureteral obstruction (UUO), Aim2 deficiency attenuated the renal injury, fibrosis, and inflammation observed in wild-type (WT) littermates. In bone marrow chimera studies, UUO induced substantially more tubular injury and IL-1β cleavage in Aim2-/- or WT mice that received WT bone marrow than in WT mice that received Aim2-/- bone marrow. Intravital microscopy of the kidney in LysM(gfp/gfp) mice 5-6 days after UUO demonstrated the significant recruitment of GFP+ proinflammatory macrophages that crawled along injured tubules, engulfed DNA from necrotic cells, and expressed active caspase-1. DNA uptake occurred in large vacuolar structures within recruited macrophages but not resident CX3CR1+ renal phagocytes. In vitro, macrophages that engulfed necrotic debris showed AIM2-dependent activation of caspase-1 and IL-1β, as well as the formation of AIM2+ ASC specks. ASC specks are a hallmark of inflammasome activation. Cotreatment with DNaseI attenuated the increase in IL-1β levels, confirming that DNA was the principal damage-associated molecular pattern in this process. Therefore, the activation of the AIM2 inflammasome by DNA from necrotic cells drives a proinflammatory phenotype that contributes to chronic injury in the kidney.

Radiographic contrast agents cause acute kidney injury (AKI), yet the underlying pathogenesis is poorly understood. Nod-like receptor pyrin containing 3-deficient (Nlrp3-deficient) mice displayed reduced epithelial cell injury and inflammation in the kidney in a model of contrast-induced AKI (CI-AKI). Unexpectedly, contrast agents directly induced tubular epithelial cell death in vitro that was not dependent on Nlrp3. Rather, contrast agents activated the canonical Nlrp3 inflammasome in macrophages. Intravital microscopy revealed diatrizoate (DTA) uptake within minutes in perivascular CX3CR1+ resident phagocytes in the kidney. Following rapid filtration into the tubular luminal space, DTA was reabsorbed and concentrated in tubular epithelial cells via the brush border enzyme dipeptidase-1 in volume-depleted but not euvolemic mice. LysM-GFP+ macrophages recruited to the kidney interstitial space ingested contrast material transported from the urine via direct interactions with tubules. CI-AKI was dependent on resident renal phagocytes, IL-1, leukocyte recruitment, and dipeptidase-1. Levels of the inflammasome-related urinary biomarkers IL-18 and caspase-1 were increased immediately following contrast administration in patients undergoing coronary angiography, consistent with the acute renal effects observed in mice. Taken together, these data show that CI-AKI is a multistep process that involves immune surveillance by resident and infiltrating renal phagocytes, Nlrp3-dependent inflammation, and the tubular reabsorption of contrast via dipeptidase-1.

BACKGROUND Protease inhibitor 9 (PI-9) is an intracellular serpin that specifically inhibits granzyme B, a cytotoxic serine protease found in the cytosolic granules of cytotoxic T lymphocytes and natural killer cells. Enhanced cortical expression of PI-9 has been observed in kidney allografts with subclinical rejection, suggesting that the tubular epithelial cell (TEC) expression of this protein may have a protective role and attenuate overt allograft rejection. METHODS AND RESULTS We demonstrate that TEC express SPI-6 protein, the murine homolog of PI-9, basally with a modest increase after cytokine exposure. Tubular epithelial cell expression of SPI-6 blocks granzyme B-mediated death because TEC from SPI-6 null kidneys have increased susceptibility to cytotoxic CD8+ cells in vitro. The role of SPI-6 was tested in a mouse kidney transplant model using SPI-6 null or wild type donor kidneys (H-2) into nephrectomized recipients (H-2). SPI-6 null kidney recipients demonstrated reduced renal function at day 8 after transplantation compared to controls (creatinine, 113±23 vs. 28±3 μmol/L; n=5; P

Cell death results in tissue damage and ultimately donor graft rejection and can occur as an active molecular process through apoptotic, necrotic and newly identified receptor interacting protein 1 and 3 kinase (RIPK1/3)-mediated necroptotic pathways. Necroptosis leads to the release of inflammatory molecules which can activate host immune cells. This pathway has yet to be studied in heart transplantation. We have found that necroptosis was induced in murine cardiac microvascular endothelial cell (MVEC) under anti-apoptotic condition following tumor necrosis factor alpha treatment. Necroptotic cell death and release of the danger molecule high mobility group box 1 (HMGB1) were inhibited by the RIPK1 inhibiting molecule necrostatin-1 and by genetic deletion of RIPK3. In addition, tissue necrosis, release of HMGB1 and graft cell infiltrate were attenuated in RIPK3 null heart allografts following transplantation. Finally, a brief sirolimus treatment markedly prolonged RIPK3 null cardiac allograft survival in allogeneic BALB/c recipients as compared to WT C57BL/6 donor grafts (95 ± 5.8 vs. 24 ± 2.6 days, p

Background: Renal ischemia reperfusion injury (IRI) leads to acute kidney injury (AKI) and the death of tubular epithelial cells (TEC). The release of high-mobility group box-1 (HMGB1) and other damage-associated molecular pattern moieties from dying cells may promote organ dysfunction and inflammation by effects on TEC. Glycyrrhizic acid (GZA) is a functional inhibitor of HMGB1, but its ability to attenuate the HMGB1-mediated injury of TEC has not been tested. Methods/Results: In vitro, hypoxia and cytokine treatment killed TEC and resulted in the progressive release of HMGB1 into the supernatant. GZA reduced the hypoxia-induced TEC death as measured by annexin-V and propidium iodide. Hypoxia increased the expression of MCP-1 and CXCL1 in TEC, which was reduced by GZA in a dose-dependent manner. Similarly, the HMGB1 activation of effector NK cells was inhibited by GZA. To test the effect of HMGB1 neutralization by GZA in vivo, mice were subjected to renal IRI. HMGB1 protein expression increased progressively in kidneys from 4 to 24 h after ischemia and was detected in tubular cells by 4 h using immunohistochemistry. GZA preserved renal function after IRI and reduced tubular necrosis and neutrophil infiltration by histological analyses and ethidium homodimer staining. Conclusions: Importantly, these data demonstrate for the first time that AKI following hypoxia and renal IRI may be promoted by HMGB1 release, which can reduce the survival of TEC and augment inflammation. Inhibition of the interaction of HMGB1 with TEC through GZA may represent a therapeutic strategy for the attenuation of renal injury following IRI and transplantation.

A hallmark feature of inflammation is the orchestrated recruitment of neutrophils from the bloodstream into inflamed tissue. Although selectins and integrins mediate recruitment in many tissues, they have a minimal role in the lungs and liver. Exploiting an unbiased in vivo functional screen, we identified a lung and liver homing peptide that functionally abrogates neutrophil recruitment to these organs. Using biochemical, genetic, and confocal intravital imaging approaches, we identified dipeptidase-1 (DPEP1) as the target and established its role as a physical adhesion receptor for neutrophil sequestration independent of its enzymatic activity. Importantly, genetic ablation or functional peptide blocking of DPEP1 significantly reduced neutrophil recruitment to the lungs and liver and provided improved survival in models of endotoxemia. Our data establish DPEP1 as a major adhesion receptor on the lung and liver endothelium and identify a therapeutic target for neutrophil-driven inflammatory diseases of the lungs.

Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naive mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)–incompatible aortic graft. The 20 S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation.

Chronic allograft injury remains the leading cause of late kidney graft loss despite improvements in immunosuppressive drugs and a reduction in acute T cell-mediated rejection. We have recently demonstrated that natural killer (NK) cells are cytotoxic to tubular epithelial cells and contribute to acute kidney ischemia-reperfusion injury. The role of NK cells in kidney allograft rejection has not been studied.A "parent to F1" kidney transplant model was used to study NK cell-mediated transplant rejection.The C57BL/6 kidneys were transplanted into fully nephrectomized CB6F1 (C57BL/6 x BALB/c) mice. Serum creatinine levels increased from baseline (18.8 ± 5.0 μmol/L to 37.2 ± 5.9 μmol/L, P0.001) at 60 days after transplantation. B6Rag-to-CB6F1Rag (B6RagxBALB/cRag) recipients, which lack T and B cells but retain NK cells, showed similar levels of kidney dysfunction 65 days after transplantation (creatinine, 33.8 ± 7.9 μmol/L vs 17.5 ± 5.1 μmol/L in nontransplant Rag mice, P0.05). Importantly, depletion of NK cells in Rag1 recipients inhibited kidney injury (24.6 ± 5.5 μmol/L, P0.05). Osteopontin, which can activate NK cells to mediate tubular epithelial cell death in vitro, was highly expressed in 60 days kidney grafts. Osteopontin null kidney grafts had reduced injury after transplantation into CB6F1 mice (17.7 ± 3.1 μmol/L, P0.001).Collectively, these data demonstrate for the first time that independent of T and B cells, NK cells have a critical role in mediating long-term transplant kidney injury. Specific therapeutic strategies that target NK cells in addition to conventional immunosuppression may be required to attenuate chronic kidney transplant injury.

Background— Although numerous signaling pathways are known to be activated in experimental cardiac hypertrophy, the molecular basis of the hypertrophic response inherent in human heart diseases remains largely unknown. Integrin-linked kinase (ILK) is a multifunctional protein kinase that physically links β-integrins with the actin cytoskeleton, suggesting a potential mechanoreceptor role. Methods and Results— Here, we show a marked increase in ILK protein levels in hypertrophic ventricles of patients with congenital and acquired outflow tract obstruction. This increase in ILK was associated with activation of the Rho family guanine triphosphatases, Rac1 and Cdc42, and known hypertrophic signaling kinases, including extracellular signal-related kinases (ERK1/2) and p70 S6 kinase. Transgenic mice with cardiac-specific expression of a constitutively active ILK (ILK S343D ) or wild-type ILK (ILK WT ) exhibited a compensated ventricular hypertrophic phenotype and displayed an activation profile of guanine triphosphatases and downstream protein kinases concordant with that seen in human hypertrophy. In contrast, transgenic mice with cardiomyocyte-restricted expression of a kinase-inactive ILK (ILK R211A ) were unable to mount a compensatory hypertrophic response to angiotensin II in vivo. Conclusions— Taken together, these results identify ILK-regulated signaling as a broadly adaptive hypertrophic response mechanism relevant to a wide range of clinical heart disease.

Human acute T-cell lymphoblastic leukemias and lymphomas (T-ALL) are commonly associated with gain-of-function mutations in Notch1 that contribute to T-ALL induction and maintenance. Starting from an expression-profiling screen, we identified c-myc as a direct target of Notch1 in Notch-dependent T-ALL cell lines, in which Notch accounts for the majority of c-myc expression. In functional assays, inhibitors of c-myc interfere with the progrowth effects of activated Notch1, and enforced expression of c-myc rescues multiple Notch1-dependent T-ALL cell lines from Notch withdrawal. The existence of a Notch1–c-myc signaling axis was bolstered further by experiments using c-myc-dependent murine T-ALL cells, which are rescued from withdrawal of c-myc by retroviral transduction of activated Notch1. This Notch1-mediated rescue is associated with the up-regulation of endogenous murine c-myc and its downstream transcriptional targets, and the acquisition of sensitivity to Notch pathway inhibitors. Additionally, we show that primary murine thymocytes at the DN3 stage of development depend on ligand-induced Notch signaling to maintain c-myc expression. Together, these data implicate c-myc as a developmentally regulated direct downstream target of Notch1 that contributes to the growth of T-ALL cells.

T-cell acute lymphoblastic leukemia (T-ALL) is a form of pediatric leukemia that is thought to be caused by approximately 12 distinct chromosomal translocations that lead to aberrant expression of as many different cellular genes. Development of novel, rational therapies against such a diverse set of mechanistic targets has thus been a formidable challenge. Recent studies, however, have identified a large fraction of T-ALL cases carrying mutations in one of these genes, Notch1, suggesting for the first time that many cases may share a common pathogenic etiology, and perhaps may allow the development of targeted therapies that benefit the majority of patients with this disease.

The incretin hormone glucagon-like peptide-1 (GLP-1) is recognized as a promising candidate for the treatment of type 2 diabetes (T2D), with one of its mimetics, exenatide (synthetic exendin-4) having already been licensed for clinical use. We seek to further improve the therapeutic efficacy of exendin-4 (Ex-4) using innovative fusion protein technology. Here, we report the production in plants a fusion protein containing Ex-4 coupled with human transferrin (Ex-4-Tf) and its characterization. We demonstrated that plant-made Ex-4-Tf retained the activity of both proteins. In particular, the fusion protein stimulated insulin release from pancreatic β-cells, promoted β-cell proliferation, stimulated differentiation of pancreatic precursor cells into insulin-producing cells, retained the ability to internalize into human intestinal cells and resisted stomach acid and proteolytic enzymes. Importantly, oral administration of partially purified Ex-4-Tf significantly improved glucose tolerance, whereas commercial Ex-4 administered by the same oral route failed to show any significant improvement in glucose tolerance in mice. Furthermore, intraperitoneal (IP) injection of Ex-4-Tf showed a beneficial effect in mice similar to IP-injected Ex-4. We also showed that plants provide a robust system for the expression of Ex-4-Tf, producing up to 37 μg prEx-4-Tf/g fresh leaf weight in transgenic tobacco and 137 μg prEx-4-Tf/g freshweight in transiently transformed leaves of N. benthamiana. These results indicate that Ex-4-Tf holds substantial promise as a new oral therapy for type 2 diabetes. The production of prEx-4-Tf in plants may offer a convenient and cost-effective method to deliver the antidiabetic medicine in partially processed plant food products.

While recent economic events have restricted opportunities for technical professionals in some geographic areas of the petroleum industry, it has boosted opportunities in others. This shift has forced many petroleum engineers to look outside their traditional marketplaces for new or continued employment or advancement opportunities and has increased the need for verification of university credentials of foreign applicants In Europe and North America, engineers rely on non-governmental, outside agencies, like the USA based Accreditation Board for Engineering and Technology Inc. (ABET), to prove their engineering programs have met established quality standards by achieving accreditation. For engineering programs in emerging markets, like the United Arab Emirates (UAE), there had been no need for accreditation beyond national organizations. However, with the accelerating global demand for professional graduates to be able to certify their credentials, several private and public universities in the UAE have just completed or are in the process of accrediting their programs. Demanding transparency and consistency, accreditation is a rigorous process whose standards force universities to refocus their output and strive for continuous improvement. One standard addressing lifelong learning opportunities, for example, requires that a program meet not only the needs of its current students but also the needs of its alumni, a group largely ignored in the past.

The late president of the United Arab Emirates, Sheikh Zayed Al Nahyan, insisted that women participate fully and equally in its growth and prosperity. In 2006, the Abu Dhabi National Oil Company (ADNOC) invited women to join their petroleum engineering educational program and study on a par with their male counterparts. ADNOC is already planning to incorporate the first female graduates into their operating companies in 2011, but are women receiving the same level of instruction as men? To help find out, the researchers initiated a study collecting data from more than 600 students over five semesters, ranging from educational background, gender, grade point average, and choice of engineering major. In addition, student and faculty surveys were conducted attempting to assess opinions about the courses, the instructors and the students.The study highlights that female employment issues are an increasingly important subject in the U.A.E. and two reasons are proposed why more women are opting to study for professional positions in the oil and gas industry.Data for student performance at the Petroleum Institute show that females are achieving almost identical academic results to males. Analysis of the wide-ranging survey data draws conclusions as to whether women will be able to compete effectively with men. The variety of attitudes to female engineers in the oil industry is presented. A significant proportion of male students reported major reservations about hiring and working with female engineers.This study demonstrates the benefits of cooperation between language and engineering faculty and concludes that fast track changes in gender diversity require commitment and changes in attitude from political leaders, oil companies and young engineers. The study will be of great interest to national oil companies, and other petroleum organizations aiming to increase use of nationals, and to all companies considering expanding opportunities for female engineers as part of a response to the changing demographics in the oil industry, commonly known as the crew change.

Renal ischemia reperfusion injury (IRI) occurs after reduced renal blood flow and is a major cause of acute injury in both native and transplanted kidneys. Studies have shown diverse cell types in both the innate and the adaptive immune systems participate in kidney IRI as dendritic cells, macrophages, neutrophils, B cells, CD4+ NK+ cells, and CD4+ T cells all contribute to this form of injury. Recently, we have found that NK cells induce apoptosis in tubular epithelial cells (TECs) and also contribute to renal IRI. However, the mechanism of NK cell migration and activation during kidney IRI remains unknown. In this study, we have identified that kidney TECs express a high level of osteopontin (OPN) in vitro and in vivo. C57BL/6 OPN-deficient mice have reduced NK cell infiltration with less tissue damage compared with wild-type C57BL/6 mice after ischemia. OPN can directly activate NK cells to mediate TEC apoptotic death and can also regulate chemotaxis of NK cells to TECs. Taken together, our study’s results indicate that OPN expression by TECs is an important factor in initial inflammatory responses that involves NK cells activity in kidney IRI. Inhibiting OPN expression at an early stage of IRI may be protective and preserve kidney function after transplantation.

From the day Edwin Drake drilled the first oil well in Titusville, Pennsylvania, in 1859, English has been the lingua franca of the petroleum industry. This standardization has allowed diverse groups from differing linguistic backgrounds to work together to get the oil to global markets efficiently and economically. The United Arab Emirates (UAE) with its large numbers of migrant workers and its vast oil reserves has certainly profited from this standardization. Founded in 1971, the UAE has become a magnet for the world’s skilled workers and has judiciously used its copious oil revenues to build a modern society. With citizens comprising less than 20% of the total residents, English has replaced native Arabic as the dominant language, not only in the oil industry but also in the daily business world of the UAE. Unified primarily by language, the migrant and local residents of the UAE, forming differing cultural groups with distinct, ethnic identities, have experienced some disagreements as the disenfranchised groups seek more inclusion and recognition. When the Abu Dhabi National Oil Company (ADNOC) opened the Petroleum Institute (PI) in 2001, its mandate was to educate Emirati young men to become engineers capable of managing ADNOC’s extensive oil fields and reserves. Since then, the PI has opened its doors to Emirati women in 2006, and Expatriate men and women in 2007. High school graduates of private and government schools in the UAE, these students are pursuing engineering studies preparing them for leadership roles within ADNOC’s Operating Companies (OpCos). This paper introduces the UAE and ADNOC’s initiative to open a local engineering school. It describes the PI’s evolving curriculum, demonstrating how it continues to adapt to meet the needs of its changing population. Clarifying some of the political and economic policies that favor Emirati students over Expatriate students and men over women, this inquiry will conclude by, first, examining the implications of the particular standards and expectations for these different groups and, second, by describing the results of a survey that explore the impact of these changes on the social cohesion among Emirati and Expatriate men and women competing for recognition in the Institute’s engineering programs and later for leadership positions within ADNOC.

One of the most important legacies of the late President, Sheikh Zayed Al Nahyan left to the people of the UAE was his insistence that women participate fully and equally in the growth and prosperity of the UAE. This paper examines the readiness of students, professors and professionals to accept women in professional roles in the oil and gas industries in the UAE. Working at the Petroleum Institute in Abu Dhabi, the researchers train both men and women to become future engineers for the Abu Dhabi National Oil Company (ADNOC). In this study, future and present professionals were asked to rate their opinions about working with women in integrated work places. To solicit these opinions, a survey of seven questions, with a place for comments, was distributed to various age groups in various job categories. Distributed in educational institutions and local petroleum societies, the survey asked participants to rate their willingness to hire, mentor, work with, promote, and work for a woman engineer. This paper analyzed a seven-statement survey. Finding that although most respondents agreed that greater numbers of women would be welcomed into the workplace, the researchers noted a stubborn minority of both men and women who did so with reservations. Looking for a plausible explanation, they concluded that stereotyping might be a motivator.described ways that included mentoring, presenting women role models, demanding academic parity, engaging in professional activities, etc. to combat stereotyping to ensure a smooth transition of more women into the work place.examined roadblocks, like [1) low number of women, 2) retaining and promoting women employees and 3) understanding differences] that companies needed to be alert to for a successful diversified culture and then listed five strategies [1) women-to-women mentoring 2) empathy vs. advice 3) anger management 4) working through problems 5) healthy environment] that must be addressed if companies want to hire and, more importantly, retain women.concluded that while programs to address the needs and attitudes of future women engineers are in place or being established, programs for future men engineers are not. This might be the first place that women and men should look for parity.

Objectives: To determine the serum and plasma level of human cardiac troponin I (cTnI) resulting from myocardial damage, we have developed a sensitive and specific one-step enzyme immunoassay to measure cardiac troponin I. Design and Methods: The COBAS® cTnI assay is a semi-automated one-step solid phase immunoassay compatible with the COBAS® Core. The assay is performed in a sandwich type format using a polyclonal goat antibody capture and two highly specific horseradish peroxidase conjugated monoclonal antibody detectors directed against different epitopes of the cTnI molecule. Calibrators were made with purified recombinant cTnI. Results: The level of cTnI was determined in 84 healthy donors with no evidence of myocardial injury, resulting in a lower limit of detection (LLD) of 0.09 μg/L. The upper reference limit (URL) of the normal reference range was calculated as 0.20 μg/L. The dynamic range of the consequent EIA was between 0.09 and 6.0 μg/L with a total assay time of 45 min. Intra-assay and inter-assay variances (CVs) were ≤4%. Cross-reactivity with fast and slow skeletal troponin I was absent in concentrations up to 2.0 mg/L. Common interferents yielded negative results in the cTnI assay. Clinical utility was confirmed by measuring the circulating serum or plasma levels of cardiac troponin I in serial samples from marathon runners, clinical samples from trauma patients, and patients presenting to the Emergency Department with complaints of chest pain. Results were further evaluated using clinical diagnosis at discharge and quantified concentrations of other cardiac markers by a Stratus® analyzer and ELISA procedures. Conclusions: Results from normal and clinical samples assayed in-house for cTnI concentrations indicate that the Spectral EIA is a highly sensitive means of quantifying cTnI levels in serum and plasma for acute cardiac syndrome. The cardiac specificity of cTnI over other well-known cardiac markers is reflected in experimental results and parallel clinical diagnosis.

We recently reported that activating mutations in the NOTCH1 receptor occur in a high percentage of primary human T-cell acute lymphoblastic leukemias (T-ALL). Withdrawal of NOTCH signals by treatment with γ-secretase inhibitors (GSI) or by transduction with a dominant-negative Mastermind-like-1 polypeptide (a specific NOTCH pathway inhibitor) induces growth arrest of many T-ALL cell lines, suggesting that NOTCH supplies signals that are needed for maintenance of growth of T-ALL cells. In order to identify downstream targets of NOTCH that mediate these effects, we performed gene expression profiling on NOTCH signaling-dependent T-ALL cell lines before and after NOTCH inhibition. Among a number of identified candidate genes was c-MYC, which was of particular interest given its importance in promoting cellular growth and its known dysregulation in a number of hematolymphoid neoplasms. c-MYC was down-regulated following NOTCH inhibition, and rapidly up-regulated following release of NOTCH inhibition, even in the presence of protein synthesis inhibitors, suggesting that it is a direct NOTCH transcriptional target. Further, a subset of murine and human T-ALL cell lines were rescued from GSI-mediated growth arrest by c-MYC-expressing retroviruses. The failure of c-MYC to rescue some NOTCH-dependent cell lines likely stems from differences in cellular context, such as collaborating oncogenic lesions and/or the stage of T cell development the cell lines recapitulate. Nevertheless, these data implicate c-MYC as a major downstream target of NOTCH in T-ALL.