Comparative proximity biotinylation implicates the small GTPase RAB18 in sterol mobilization and biosynthesis.

Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18 interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor complex. Twelve of these 28 interactions are supported by prior reports, and we have directly validated novel interactions with SEC22A, TMCO4, and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites, interactors included groups of microtubule/membrane-remodeling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP is a Δ8-Δ7 sterol isomerase, and ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We found that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Furthermore, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated or in which ORP2 expression is disrupted. Our data demonstrate that guanine nucleotide exchange factor-dependent Rab interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.

Combining steady state and temperature jump IR spectroscopy to investigate the allosteric effects of ligand binding to dsDNA.

Changes in the structural dynamics of double stranded (ds)DNA upon ligand binding have been linked to the mechanism of allostery without conformational change, but direct experimental evidence remains elusive. To address this, a combination of steady state infrared (IR) absorption spectroscopy and ultrafast temperature jump IR absorption measurements has been used to quantify the extent of fast (∼100 ns) fluctuations in (ds)DNA·Hoechst 33258 complexes at a range of temperatures. Exploiting the direct link between vibrational band intensities and base stacking shows that the absolute magnitude of the change in absorbance caused by fast structural fluctuations following the temperature jump is only weakly dependent on the starting temperature of the sample. The observed fast dynamics are some two orders of magnitude faster than strand separation and associated with all points along the 10-base pair duplex d(GCATATATCC). Binding the Hoechst 33258 ligand causes a small but consistent reduction in the extent of these fast fluctuations of base pairs located outside of the ligand binding region. These observations point to a ligand-induced reduction in the flexibility of the dsDNA near the binding site, consistent with an estimated allosteric propagation length of 15 Å, about 5 base pairs, which agrees well with both molecular simulation and coarse-grained statistical mechanics models of allostery leading to cooperative ligand binding.

Development of hepatic pathology in GBV-B-infected red-bellied tamarins (Saguinus labiatus).

GB virus B (GBV-B) is a new world monkey-associated flavivirus used to model acute hepatitis C virus (HCV) infection. Critical for evaluation of antiviral or vaccine approaches is an understanding of the effect of HCV on the liver at different stages of infection. In the absence of longitudinal human tissue samples at defined time points, we have characterized changes in tamarins. As early as 2 weeks post-infection histological changes were noticeable, and these were established in all animals by 6 weeks. Despite high levels of liver-associated viral RNA, there was reversal of hepatic damage on clearance of peripheral virus though fibrosis was demonstrated in four tamarins. Notably, viral RNA burden in the liver dropped to near undetectable or background levels in all animals which underwent a second viral challenge, highlighting the efficacy of the immune response in removing foci of replication in the liver. These data add to the knowledge of GBV-B infection in New World primates which can offer attractive systems for the testing of prophylactic and therapeutic treatments and the evaluation of their utility in preventing or reversing liver pathology.

Intravenous pulse methylprednisolone for induction of remission in severe ANCA associated Vasculitis: a multi-center retrospective cohort study.

BACKGROUND: Intravenous pulse methylprednisolone (MP) is commonly included in the management of severe ANCA associated vasculitis (AAV) despite limited evidence of benefit. We aimed to evaluate outcomes in patients who had, or had not received MP, along with standard therapy for remission induction in severe AAV. METHODS: We retrospectively studied 114 consecutive patients from five centres in Europe and the United States with a new diagnosis of severe AAV (creatinine > 500 µmol/L or dialysis dependency) and that received standard therapy (plasma exchange, cyclophosphamide and high-dose oral corticosteroids) for remission induction with or without pulse MP between 2000 and 2013. We evaluated survival, renal recovery, relapses, and adverse events over the first 12 months. RESULTS: Fifty-two patients received pulse MP in addition to standard therapy compared to 62 patients that did not. There was no difference in survival, renal recovery or relapses. Treatment with MP associated with higher risk of infection during the first 3 months (hazard ratio (HR) 2.7, 95%CI [1.4-5.3], p = 0.004) and higher incidence of diabetes (HR 6.33 [1.94-20.63], p = 0.002), after adjustment for confounding factors. CONCLUSIONS: The results of this study suggest that addition of pulse intravenous MP to standard therapy for remission induction in severe AAV may not confer clinical benefit and may be associated with more episodes of infection and higher incidence of diabetes.

Rituximab for treatment of severe renal disease in ANCA associated vasculitis.

BACKGROUND: Rituximab (RTX) is approved for remission induction in ANCA associated vasculitis (AAV). However, data on use of RTX in patients with severe renal disease is lacking. METHODS: We conducted a retrospective multi-center study to evaluate the efficacy and safety of RTX with glucocorticoids (GC) with and without use of concomitant cyclophosphamide (CYC) for remission induction in patients presenting with e GFR less than 20 ml/min/1.73 m(2). We evaluated outcomes of remission at 6 months (6 M), renal recovery after acute dialysis at diagnosis, e-GFR rise at 6 M, patient and renal survival and adverse events. RESULTS: A total 37 patients met the inclusion criteria. The median age was 61 years. (55-73), 62 % were males, 78 % had new diagnosis and 59 % were MPO ANCA positive. The median (IQR) e-GFR at diagnosis was 13 ml/min/1.73 m(2) (7-16) and 15 required acute dialysis. Eleven (30 %) had alveolar hemorrhage. Twelve (32 %) received RTX with GC, 25 (68 %) received RTX with GC and CYC and seventeen (46 %) received plasma exchange. The median (IQR) follow up was 973 (200-1656) days. Thirty two of 33 patients (97 %) achieved remission at 6 M and 10 of 15 patients (67 %) requiring dialysis recovered renal function. The median prednisone dose at 6 M was 6 mg/day. The mean (SD) increase in e-GFR at 6 months was 14.5 (22) ml/min/m(2). Twelve patients developed ESRD during follow up. There were 3 deaths in the first 6 months. When stratified by use of concomitant CYC, there were no differences in baseline e GFR, use of plasmapheresis, RTX dosing regimen or median follow up days between the groups. No differences in remission, renal recovery ESRD or death were observed. CONCLUSIONS: This study of AAV patients with severe renal disease demonstrates that the outcomes appear equivalent when treated with RTX and GC with or without concomitant CYC.

CD4+CD28- T-cell expansions in ANCA-associated vasculitis and association with arterial stiffness: baseline data from a randomised controlled trial.

BACKGROUND: Cardiovascular risk is increased in the anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitides (AAV). CD4+CD28- T cells are expanded in patients with AAV who are seropositive for cytomegalovirus (CMV), and are associated with increased mortality. CMV seropositivity in other conditions is associated with arterial stiffness, a marker of cardiovascular risk. We assessed whether CD4+CD28- T cells in CMV seropositive patients with AAV are associated with arterial stiffness and whether treatment with valaciclovir reduces this cell population. METHODS: In this open-label phase 2 trial, patients were randomised (1:1) by computer to valaciclovir (8 g daily) or no additional treatment for 6 months. Primary outcome was proportion of patients with CMV reactivation. Arterial stiffness (carotid to femoral pulse wave velocity [PWV]) was measured and peripheral blood CD4+CD28- T cells analysed by flow cytometry at baseline and 6 months. CD4+CD28- T-cell interferon γ (IFNγ) secretion was stimulated with CMV lysate. Data are presented as median (IQR). Between-group differences were tested by Mann-Whitney U test and correlations by Spearman's rank. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01633476. FINDINGS: Baseline data are presented from the first 28 patients enrolled, with 6 months' follow-up completed in five treatment and six control patients. More CD4+CD28- than CD4+CD28+ T cells expressed T-bet (83·5% [47·5-89·9] vs 12·9 [4·7-22·2], p<0·0001) and secreted IFNγ after stimulation (14·3% [8·4-26·0] vs 0·8 [0·2-1·3], p<0·0001). CX3CR1, a cell surface marker whose expression is associated with endothelial dysfunction, was only expressed on CD4+CD28- T cells. The proportion of CD4+CD28- T cells correlated with PWV (r=0·408, p=0·035). At 6 months, reduction in proportion of CD4+CD28- T cells was greater in the treatment than in the control group (-1·8% [-5·2 to -0·6] vs 0·5 [-0·8 to 2·5], p=0·044). INTERPRETATION: These preliminary results suggest that CD4+CD28- T cells in AAV are proinflammatory cells with high expression of the fractalkine receptor CX3CR1 that has been implicated in endothelial dysfunction. This cell population is associated with arterial stiffness, and its expansion is attenuated with valaciclovir treatment. This research has important implications, because cardiovascular disease is a major cause of mortality in AAV. FUNDING: Wellcome Trust, Vasculitis UK.

Treatment of severe renal disease in ANCA positive and negative small vessel vasculitis with rituximab.

BACKGROUND/AIMS: Rituximab and glucocorticoids are a non-inferior alternative to cyclophosphamide and glucocorticoid therapy for induction of remission in antineutrophil cytoplasmic antibody (ANCA) associated vasculitis (AAV) patients with moderate renal disease. The efficacy and safety of this approach in patients with severe renal impairment are unknown. We report the outcomes and safety profile of rituximab and glucocorticoid therapy for induction of remission in patients with AAV and ANCA-negative vasculitis presenting with severe renal disease. METHODS: A multicenter, retrospective, cohort study was conducted between 2005 and 2014. Patients with new or relapsing disease with an estimated glomerular filtration rate (eGFR) of ≤20 ml/min/1.73 m(2) treated with rituximab and glucocorticoid induction with or without plasmapheresis were included. Fourteen patients met the inclusion criteria. The primary outcomes were rate of remission and dialysis independence at 6 months. The secondary outcomes were eGFR at 6 months, end-stage renal disease (ESRD), survival rates and adverse events. RESULTS: All patients were Caucasian, and 57% were male. The mean eGFR was 12 ml/min/1.73 m(2) at diagnosis. All patients achieved remission with a median time to remission of 55 days. Seven patients required dialysis at presentation of which 5 patients recovered renal function and discontinued dialysis by 6-month follow-up. The mean eGFR for the 11 patients without ESRD who completed 6-month follow-up was 33 ml/min/1.73 m(2). Four patients ultimately developed ESRD, and one died during the follow-up period. CONCLUSION: Patients with AAV and severe renal disease achieve high rates of remission and dialysis independence when treated with rituximab and glucocorticoids without cyclophosphamide.

Changes in immune cell populations in the periphery and liver of GBV-B-infected and convalescent tamarins (Saguinus labiatus).

Flaviviruses related to hepatitis C virus (HCV) in suitable animal models may provide further insight into the role that cellular immunity contributes to spontaneous clearance of HCV. We characterised changes in lymphocyte populations in tamarins with an acute GBV-B infection, a hepatitis virus of the flaviviridae. Major immune cell populations were monitored in peripheral and intra-hepatic lymphocytes at high viraemia or following a period when peripheral virus was no longer detected. Limited changes in major lymphocyte populations were apparent during high viraemia; however, the proportions of CD3(+) lymphocytes decreased and CD20(+) lymphocytes increased once peripheral viraemia became undetectable. Intrahepatic lymphocyte populations increased at both time points post-infection. Distinct expression patterns of PD-1, a marker of T-cell activation, were observed on peripheral and hepatic lymphocytes; notably there was elevated PD-1 expression on hepatic CD4(+) T-cells during high viraemia, suggesting an activated phenotype, which decreased following clearance of peripheral viraemia. At times when peripheral vRNA was not detected, suggesting viral clearance, we were able to readily detect GBV-B RNA in the liver, indicative of long-term virus replication. This study is the first description of changes in lymphocyte populations during GBV-B infection of tamarins and provides a foundation for more detailed investigations of the responses that contribute to the control of GBV-B infection.

Local partnerships for community assessment and planning.

PURPOSE: Community assessment is a core function of public health. National and state policies encourage local health departments (LHDs) to engage local partners in conducting community assessments. This study examined the prevalence, characteristics, and effectiveness of community assessment partnerships between LHDs and other organizations. METHODS: LHDs in Wisconsin completed a 2-stage, cross-sectional survey. A subset analysis of community assessment partnerships was conducted using descriptive, bivariate, and multivariate statistical methods. RESULTS: Ninety percent of LHDs reported a partnership focused on community assessment (n = 69). Sixty-six percent of community assessment partnerships had existed for three or more years, and all of these had implemented plans (n = 43). Community assessment partnerships were more likely than partnerships focused on other issues to have formed because of a mandate, to include many partners, and to receive some forms of financial support from the LHD. Partnerships focused on community assessment were no more likely to be effective than other types of partnerships. CONCLUSIONS: LHDs and community partners realize mutual benefits from collaborating on community assessment. Successful community assessment partnerships can be supported by building competencies in the public health workforce and sustaining partnerships for substantial periods of time.