Search

Objectives: ANCA-associated vasculitis (AAV) is a rare autoimmune disorder that commonly involves the kidney. Early identification of kidney involvement, assessing treatment-response and predicting outcome are important clinical challenges. Here, we assessed the potential utility of interval kidney biopsy in AAV., Methods: In a tertiary referral centre with a dedicated vasculitis service, we identified patients with AAV who had undergone interval kidney biopsy, defined as a repeat kidney biopsy (following an initial biopsy showing active AAV) undertaken to determine the histological response in the kidney following induction immunosuppression. We analysed biochemical, histological and outcome data, including times to kidney failure and death for all patients., Results: We identified 57 patients with AAV who underwent at least one interval kidney biopsy (59 interval biopsies in total; median time to interval biopsy ∼130 days). Of the 59 interval biopsies performed, 24 (41%) patients had clinically suspected active disease at time of biopsy which was confirmed histologically in only 42% of cases; 35 (59%) patients were in clinical disease-remission, and this was correct in 97% of cases. The clinician's impression was incorrect in one in four patients. Hematuria at interval biopsy did not correlate with histological activity. Interval biopsy showed fewer acute lesions and more chronic damage compared with initial biopsy and led to immunosuppressive treatment-change in 75% (44/59) of patients. Clinical risk prediction tools tended to operate better using interval biopsy data., Conclusion: Interval kidney biopsy is useful for determining treatment-response and subsequent disease management in AAV. It may provide better prognostic information than initial kidney biopsy and should be considered for inclusion into future clinical trials and treatment protocols for patients with AAV., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Objective: ANCA-associated vasculitis (AAV) is a small vessel vasculitis that commonly presents in the elderly. However, there are few long-term outcome data for these patients. Here, we assessed long-term outcomes in a single-centre cohort of elderly patients with AAV. Additionally, we tested whether a pre-morbid frailty score could aid prognosis., Methods: Using a prospectively-compiled dataset, we investigated patients over the age of 65 who presented with AAV between 2005 and 2017 to a regional vasculitis centre. We used a Cox model to determine the factors associated with mortality. We also compared outcomes in pre-specified subgroups stratified by baseline frailty score, ANCA serotype and induction immunosuppression (with cyclophosphamide, rituximab or mycophenolate mofetil used as the main glucocorticoid-sparing agent)., Results: 83 patients were included in the study and were followed for a median of 1203 days. Median age was 74 years (range 65-92). Two- and five-year survival in the overall cohort were 83% (95% CI 75, 92%) and 75% (95% CI 65, 86%), respectively. The median cumulative dose of oral prednisolone was 2030 mg during the first three months. Only one patient received intravenous glucocorticoids. Age, frailty score and CRP at presentation were independently associated with mortality; all deaths occurred in patients aged over 75 at presentation. Patients treated with a cyclophosphamide-based induction regimen tended to be younger than those treated with rituximab or mycophenolate mofetil. Survival was better in the cyclophosphamide-treated group., Conclusion: In the contemporary era, the overall prognosis of AAV in elderly patients is good. Baseline frailty associates with disease outcomes including mortality. A low-dose glucocorticoid regimen (avoiding intravenous methylprednisolone) can be used to treat AAV effectively in elderly patients., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Study Objectives: Depressive symptoms following adenotonsillectomy (AT) relative to controls were examined in children with obstructive sleep apnea syndrome (OSAS)., Methods: The Childhood Adenotonsillectomy Trial (CHAT) multisite study examined the impact of AT in 453 children aged 5 to 9.9 years with polysomnographic evidence of OSAS without prolonged desaturation, randomized to early adenotonsillectomy (eAT) or watchful waiting with supportive care (WWSC). One hundred seventy-six children (eAT n = 83; WWSC n = 93) with complete evaluations for depressive symptomatology between baseline and after a 7-month intervention period were included in this secondary analysis., Results: Exact binomial test assessed proportion of depressive symptomatology relative to norms, while effects of AT and OSAS resolution were assessed through linear quantile mixed-models. Treatment group assignment did not significantly impact depression symptoms, although self-reported depression symptoms improved over time (p < 0.001). Resolution of OSAS symptoms demonstrated a small interaction effect in an unexpected direction, with more improvement in parent ratings of anxious/depressed symptoms for children without resolution (p = 0.030). Black children reported more severe depressive symptoms (p = 0.026) and parents of overweight/obese children reported more withdrawn/depressed symptoms (p = 0.004). Desaturation nadir during sleep was associated with self-report depressed (r = -0.17, p = 0.028), parent-reported anxious/depressed (r = -0.15, p = 0.049), and withdrawn/depressed (r = -0.24, p = 0.002) symptoms., Conclusions: Increased risk for depressed and withdrawn/depressed symptoms was detected among children with OSAS, and different demographic variables contributed to risk in self-reported and parent-reported depression symptoms. Arterial oxygen desaturation nadir during sleep was strongly associated with depressed symptoms. However, despite improvements in child-reported depressed symptoms over time, changes were unrelated to either treatment group or OSAS resolution status., Trials Registration: Childhood Adenotonsillectomy Study for Children with OSAS (CHAT), https://clinicaltrials.gov/show/NCT00560859, NCT00560859.

Background: Neuraminidase (NA) inhibitors are the recommended antiviral medications for influenza treatment. However, their therapeutic efficacy can be compromised by NA changes that emerge naturally and/or following antiviral treatment. Knowledge of which molecular changes confer drug resistance of influenza A(H7N9) viruses (group 2NA) remains sparse., Methods: Fourteen amino acid substitutions were introduced into the NA of A/Shanghai/2/2013(H7N9). Recombinant N9 (recN9) proteins were expressed in a baculovirus system in insect cells and tested using the Centers for Disease Control and Prevention standardized NA inhibition (NI) assay with oseltamivir, zanamivir, peramivir, and laninamivir. The wild-type N9 crystal structure was determined in complex with oseltamivir, zanamivir, or sialic acid, and structural analysis was performed., Results: All substitutions conferred either reduced or highly reduced inhibition by at least 1 NA inhibitor; half of them caused reduced inhibition or highly reduced inhibition by all NA inhibitors. R292K conferred the highest increase in oseltamivir half-maximal inhibitory concentration (IC50), and E119D conferred the highest zanamivir IC50. Unlike N2 (another group 2NA), H274Y conferred highly reduced inhibition by oseltamivir. Additionally, R152K, a naturally occurring variation at the NA catalytic residue of A(H7N9) viruses, conferred reduced inhibition by laninamivir., Conclusions: The recNA method is a valuable tool for assessing the effect of NA changes on drug susceptibility of emerging influenza viruses., (Published by Oxford University Press for the Infectious Diseases Society of America 2017. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Objectives: The childhood obstructive sleep apnea syndrome (OSAS) is associated with behavioral abnormalities. Studies on the effects of OSAS treatment on behavior are conflicting, with few studies using a randomized design. Further, studies may be confounded by the inclusion of behavioral outcome measures directly related to sleep. The objective of this study was to determine the effect of adenotonsillectomy on behavior in children with OSAS. We hypothesized that surgery would improve behavioral ratings, even when sleep symptom items were excluded from the analysis., Methods: This was a secondary analysis of Child Behavior Checklist (CBCL) data, with and without exclusion of sleep-specific items, from the Childhood Adenotonsillectomy Trial (CHAT). CBCL was completed by caregivers of 380 children (7.0+1.4 [range 5-9] years) with OSAS randomized to early adenotonsillectomy (eAT) versus 7 months of watchful waiting with supportive care (WWSC)., Results: There was a high prevalence of behavioral problems at baseline; 16.6% of children had a Total Problems score in the clinically abnormal range. At follow-up, there were significant improvements in Total Problems (p < .001), Internalizing Behaviors (p = .04), Somatic Complaints (p = .01), and Thought Problems (p = .01) in eAT vs. WWSC participants. When specific sleep-related question items were removed from the analysis, eAT showed an overall improvement in Total (p = .02) and Other (p = .01) problems. Black children had less improvement in behavior following eAT than white children, but this difference attenuated when sleep-related items were excluded., Conclusions: This large, randomized trial showed that adenotonsillectomy for OSAS improved parent-rated behavioral problems, even when sleep-specific behavioral issues were excluded from the analysis., (© Sleep Research Society 2017. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.)

Motivation: Post-transcriptional and co-transcriptional regulation is a crucial link between genotype and phenotype. The central players are the RNA-binding proteins, and experimental technologies [such as cross-linking with immunoprecipitation- (CLIP-) and RIP-seq] for probing their activities have advanced rapidly over the course of the past decade. Statistically robust, flexible computational methods for binding site identification from high-throughput immunoprecipitation assays are largely lacking however., Results: We introduce a method for site identification which provides four key advantages over previous methods: (i) it can be applied on all variations of CLIP and RIP-seq technologies, (ii) it accurately models the underlying read-count distributions, (iii) it allows external covariates, such as transcript abundance (which we demonstrate is highly correlated with read count) to inform the site identification process and (iv) it allows for direct comparison of site usage across cell types or conditions., Availability and Implementation: We have implemented our method in a software tool called Piranha. Source code and binaries, licensed under the GNU General Public License (version 3) are freely available for download from http://smithlab.usc.edu., Contact: andrewds@usc.edu, Supplementary Information: Supplementary data available at Bioinformatics online.

Summary: We report on a major new version of the RMAP software for mapping reads from short-read sequencing technology. General improvements to accuracy and space requirements are included, along with novel functionality. Included in the RMAP software package are tools for mapping paired-end reads, mapping using more sophisticated use of quality scores, collecting ambiguous mapping locations and mapping bisulfite-treated reads., Availability: The applications described in this note are available for download at http://www.cmb.usc.edu/people/andrewds/rmap and are distributed as Open Source software under the GPLv3.0. The software has been tested on Linux and OS X platforms., Contact: andrewds@usc.edu; mzhang@cshl.edu

Loss-of-function by means of RNA interference in cultured human cells enables rapid pathway dissection on a genome-scale. Improved siRNA design and key validation protocols are required to eliminate falsely identified phenotypes resulting from potential off-target consequences. Here, we demonstrate a validation strategy involving several steps for verifying cell death phenotypes revealed during loss-of-function screening. First, from a set of 45 novel human genes we identified gene candidates that, when silenced, induce apoptosis in cultured HeLa cells. For those candidates, we performed more extensive validation with multiple effective siRNAs. In addition, we designed rescue experiments involving candidate genes delivered exogenously and containing silent mutations in the siRNA target regions. Rescue of the observed knockdown phenotype demonstrated an original and more stringent validation of the siRNA's selectivity and the phenotype specificity for the target gene. As a result, our data reveals an anti-apoptotic function for novel human breast adenocarcinoma marker BC-2, adding new depth to BC-2's description as a putative tumor marker involved in cancer related pathways.

An important aim in the post-sequencing age of functional genomics is to translate gene sequences into protein functions. This shift of focus is particularly necessary for a very large number of human genes, referred to as novel genes, where we have no or very rudimentary information about their biochemical functions. Recently, a new method for investigating human gene functions using small interfering RNA molecules (siRNAs) has become available. siRNAs are powerful reagents for post-transcriptional silencing, where mRNA targeted by the siRNAs is degraded in vivo and the level of the encoded protein is reduced. However, the lack of antibodies against proteins encoded by novel genes restricts the general value of siRNAs as functional tools, as only the mRNA levels can be measured for these genes. We report a method that combines measurements of protein levels in cell culture for novel, exogenously expressed genes, with parallel measurements of the endogenous mRNA levels of the same genes. We find that this combinatorial approach correctly predicts siRNAs that efficiently reduce mRNA and protein levels in cultured cells. Furthermore, this method identifies proteins that have a slow turnover, which weakens the value of the RNA interference method as a tool for functional studies of such genes. The described method should prove to be valuable for large-scale functional studies of novel human genes.

We have undertaken a retrospective study of antibody deficient patients, with and without lymphoma, and assessed the ability of specific polymerase chain reaction (PCR) primers to determine if the detection of clonal lymphocyte populations correlates with clinical and immunohistochemical diagnosis of lymphoma. We identified 158 cases with antibody deficiency presenting during the past 20 years. Paraffin-embedded biopsy specimens or slides were available for analysis in a cohort of 34 patients. Of these patients, 29 had common variable immunodeficiency, one X-linked agammaglobulinaemia, one X-linked immunoglobulin deficiency of uncertain cause and three isolated IgG subclass deficiency. We have confirmed that lymphoma in antibody deficiency is predominantly B cell in origin. Clonal lymphocyte populations were demonstrated in biopsies irrespective of histology (16/19 with lymphoma and 11/15 without). Isolated evidence of clonality in biopsy material is therefore an insufficient diagnostic criterion to determine malignancy. Furthermore, our data suggest that clonal expansions are rarely the result of Epstein-Barr virus-driven disease.

In this study we examined the cytokine production by T cells and TCRVbeta subsets in peripheral blood (PB) and synovial fluid (SF) from six RA patients and PB from 10 normal subjects, using three-colour flow cytometry. In two RA subjects we assessed T cell clonality by RT PCR using TCRBV family-specific primers and analysed the CDR3 (complementarity determining region 3) length by GeneScan analysis. A high percentage of IFN-gamma- and IL-2- producing cells was observed among the PB T cells in both the RA patients and normal controls and among the SF T cells in RA patients. In contrast, the percentage of T cells producing IL-4 and IL-5 was small among PB T cells in both RA patients and normal controls and among SF T cells in RA patients. There was no significant difference in the production of IFN-gamma, IL-2 and IL-5 between the two compartments (PB and SF); however, there were significantly more IL-4-producing cells in SF. Molecular analysis revealed clonal expansions of four TCRBV families in SF of two of the RA patients studied: TCRBV6.7, TCRBV13.1 and TCRBV22 in one and TCRBV6.7, TCRBV21.3 and TCRBV22 in the second. These expansions demonstrated cytokine expression profiles that differed from total CD3+ cells, implying that T cell subsets bearing various TCR-Vbeta families may have the potential to modulate the immune response in RA patients.

T cells are thought to play an important regulatory role in asthma, but little is known about the T cell repertoire of the human lung or whether asthma is associated with any specific repertoire changes. Flow cytometry and MoAbs to TCR VB (TCRBV) families were used to quantify bronchoalveolar lavage (BAL) and blood T cells from normal and atopic individuals. Clonality was then assessed by polymerase chain reaction (PCR) amplification of cDNA and gene scanning using consensus and family-specific TCRBV primers and confirmed by sequence analysis. In addition, blood and BAL T cell populations were studied pre- and post-allergen challenge in four patients with allergic asthma. The majority of TCRBV families detected in blood by MoAb staining were also represented in BAL. While differences between BAL and blood populations were evident in each individual studied, these differences were not consistent between individuals or between CD4+ and CD8+ T cell subpopulations. These results are in broad agreement with other published studies, but in contrast to previous work we found a consistent difference between TCRBV7 family usage in blood and BAL in all individuals studied, and a consistently increased proportion of CD4+ BAL T cells bearing BV5S2/3 in asthmatics only. After allergen challenge, the pattern of TCRBV gene usage was largely unchanged as judged by flow cytometry. Gene scanning of PCR products generated from consensus VB primers revealed polyclonal lymphocyte populations in blood and BAL from all seven atopic individuals: in one normal tested polyclonal populations were found in blood and oligoclonal populations in BAL. Selected families amplified with family-specific primers BV5S2/3, BV6 and BV7 (chosen because of their predominance in BAL compared with blood) were more variable and revealed predominant polyclonal populations in blood and polyclonal or oligoclonal populations in BAL. In one asthmatic patient a clonal BV5S2 family was found in BAL. Following allergen challenge there were no significant changes in polyclonality/oligoclonality/clonality in three cases, but in one case a clonal BV5S2 population was found after challenge, that had not been evident beforehand. The lung T cell repertoire is thus broadly representative of blood T cells, but shows population differences that may result from response to persistent exposure to airborne antigens common to normal and atopic individuals. Oligoclonal TCRBV family expansion appears to be primarily lung-specific but independent of atopic asthma, although our challenge data in one case support the concept that clonal populations may follow local allergen challenge. These data are consistent with selection and amplification of specific T cell families in the lung in response to local antigenic exposure.

This report documents the presence of clonal gamma delta T-cell receptor (TcR) population in the blood of a patient who presented with an arthropathy of undetermined cause, leucopenia and splenomegaly. There was no evidence for lymphoid malignancy clinically or at post-mortem. The phenotype and genotype of the clonal T-cell population were not associated with the predominant TcR delta rearrangement found in peripheral blood gamma delta cells, but were similar to those found in gamma delta TcR cells infiltrating rheumatoid synovium. The data indicate the presence of a monoclonal population of gamma delta cells TcR cells which in the face of continued immunosuppression behaved benignly. The case may represent a cytomorphologically atypical example of the large granular lymphocytes, neutropenia and arthropathy syndrome/lymphoproliferative disease of granular lymphocytes and, although the patient's clinical features were not 'classical', rheumatoid arthritis (RA) may have been the underlying primary disorder.

We compared the diagnostic and prognostic utility of phospholipase A (PLA; EC 3.1.1.4) for acute pancreatitis with that of amylase and lipase by analysis of sera from 151 consecutive patients presenting with abdominal pain in whom assays of serum amylase and (or) lipase had been ordered. We determined the diagnostic accuracy for both the initial and the peak enzyme activities. Maximal diagnostic accuracy obtained for the initial activities of amylase, lipase, and PLA was 0.83, 0.83, and 0.76 at cutoff values of 650, 650, and 41 U/L, respectively. Use of peak enzyme activities showed maximal diagnostic accuracy of 0.85, 0.86, and 0.73 at cutoff values of 650, 1050, and 42 U/L, respectively. Receiver-operator characteristic curve analysis revealed the diagnostic performance of amylase and lipase to be similar, whereas that of PLA was almost random and not incremental. As with amylase and lipase, PLA activities in sera showed no relation to patients' survival; three patients who died after an attack of acute pancreatitis failed to demonstrate the dramatic increases in PLA activity previously described. We conclude that assessing the severity of acute pancreatitis by using enzyme activities still remains problematical. Measurements of amylase or lipase activities provide similar diagnostic discrimination when appropriate cutoff values are used and remain the methods of choice for diagnosis of acute pancreatitis.

We investigated the effect of reticulocytosis on the lactate dehydrogenase (LD; EC 1.1.1.27) isoenzyme LD1/LD2 ratio in patients with and without evidence of hemolytic disease. Analysis of sera from patients with reticulocytosis and in vivo hemolysis showed a mean LD1/LD2 ratio of 0.92 compared with a ratio of 0.69 in patients with in vivo hemolysis and normal reticulocyte counts. Determination of LD isoenzymes in erythrocyte lysate revealed significantly increased LD1/LD2 ratios for patients with marked reticulocytosis compared with those for patients with normal-to-minimal increases in reticulocytes. Finally, separation of mature erythrocytes and reticulocytes by flow cytometry revealed marked differences in the LD1/LD2 isoenzyme distribution between these two cell types. The ability of hemolysis to cause a "flipped" LD1/LD2 ratio is dependent on the proportion of the hemolyzed cells that are reticulocytes.