Publications

Aire controls immunologic tolerance by inducing a battery of thymic transcripts encoding proteins characteristic of peripheral tissues. Its unusually broad effect is achieved by releasing RNA polymerase II paused just downstream of transcriptional start sites. We explored Aire's collaboration with the bromodomain-containing protein, Brd4, uncovering an astonishing correspondence between those genes induced by Aire and those inhibited by a small-molecule bromodomain blocker. Aire:Brd4 binding depended on an orchestrated series of posttranslational modifications within Aire's caspase activation and recruitment domain. This interaction attracted P-TEFb, thereby mobilizing downstream transcriptional elongation and splicing machineries. Aire:Brd4 association was critical for tolerance induction, and its disruption could account for certain point mutations that provoke human autoimmune disease. Our findings evoke the possibility of unanticipated immunologic mechanisms subtending the potent antitumor effects of bromodomain blockers.

Reducing geographic barriers to tuberculosis (TB) care is a priority in high-burden countries where patients frequently initiate, but do not complete, the multi-day TB evaluation process. Using routine cross-sectional study from six primary-health clinics in rural Uganda from 2009 to 2012, we explored whether geographic barriers affect completion of TB evaluation among adults with unexplained chronic cough. We measured distance from home parish to health center and calculated individual travel time using a geographic information systems technique incorporating roads, land cover, and slope, and measured its association with completion of TB evaluation. In 264,511 patient encounters, 4,640 adults (1.8%) had sputum smear microscopy ordered; 2,783 (60%) completed TB evaluation. Median travel time was 68 minutes for patients with TB examination ordered compared with 60 minutes without (P < 0.010). Travel time differed between those who did and did not complete TB evaluation at only one of six clinics, whereas distance to care did not differ at any of them. Neither distance nor travel time predicted completion of TB evaluation in rural Uganda, although limited detail in road and village maps restricted full implementation of these mapping techniques. Better data are needed on geographic barriers to access clinics offering TB services to improve TB diagnosis.

BACKGROUND & AIMS

Although patients infected by genotype 1b hepatitis C virus (HCV) with Q(70) and/or M(91)core gene mutations have an almost five-fold increased risk of developing hepatocellular carcinoma (HCC) and increased insulin resistance, the absence of a suitable experimental system has precluded direct experimentation on the effects of these mutations on cellular gene expression.

METHODS

HuH7 cells were treated long-term with human serum to induce differentiation and to produce a model system for testing high-risk and control HCV. For clinical validation, profiles of infected cells were compared to each other and to those of liver biopsies of patients with early-stage HCV-related cirrhosis followed prospectively for up to 23 years (n=216).

RESULTS

Long-term culture in human serum produced growth-arrested, hepatocyte-like cells whose gene profile overlapped significantly with that of primary human hepatocytes. High-risk (Q(70)/M(91)) and control (R(70)/L(91)) viruses had dramatically different effects on gene expression of these cells. The high-risk virus enhanced expression of pathways associated with cancer and type II diabetes, while the control virus enhanced pathways associated with oxidative phosphorylation. Of special clinical relevance, the transcriptome of cells replicating the high-risk virus correlated significantly with an HCC high-risk profile in patients (Bonferroni-corrected p=0.03), whereas no such association was observed for non-HCC-related clinical outcomes.

CONCLUSIONS

The cell-based system allowed direct head-to-head comparison of HCV variants, and provided experimental support for previous clinical data indicating an oncogenic effect of core gene mutations. This simple experimental system distinguished HCV variants and will enable future mechanistic analysis and exploration of interventional approaches.

INTRODUCTION

There are several methods to induce ventricular fibrillation (VF) during defibrillation threshold (DFT) testing. Delivering a shock at a critical time during the T wave (T-shock) is the conventional approach, while delivering a constant direct current voltage (DC stim) from the implantable cardioverter defibrillator is an alternative method. Only a few reports compare VF induction methods. The purpose of this study was to evaluate the effects and safety of DC stim versus T-shock.

METHODS

We retrospectively investigated 414 consecutive patients undergoing DFT testing. We compared the two groups (DC stim and T-shock) with respect to clinical characteristics, electrocardiogram (ECG) changes, and complications.

RESULTS

Ventricular arrhythmia, including ventricular tachycardia (VT) and VF, was induced by DC stim in 93 patients or T-shock in 321 patients. No more than three attempts were performed during one procedure. There was no significant difference in the baseline ECG, induced tachycardia cycle length (TCL), or complications between the two groups. However, the induced TCL was significantly shorter than the clinical TCL regardless of induction method (P = 0.001). Five patients suffered major complications (i.e., electromechanical dissociation or incessant VT). A history of atrial fibrillation was significantly greater in patients with major complications than the others (80% vs 24%, P = 0.004), and was an independent predictor on multivariate analysis.

CONCLUSIONS

There is no significant difference in induced TCL or complications between the DC stim and T-shock. The induced TCL is significantly shorter than clinical TCL regardless of induction method.

BACKGROUND

Lysosomal acid lipase (LAL), encoded by the LIPA gene, catalyzes the intracellular hydrolysis of cholesteryl esters and triglycerides in hepatocytes and macrophages. LIPA defects cause accumulation of these lipids in lysosomes. LAL deficiency (LAL D) presents and progresses as a continuum with dyslipidemia, hepatomegaly, and liver fibrosis.

OBJECTIVE

To improve the understanding of the genetic basis of LAL D, an underappreciated cause of dyslipidemia and cirrhosis, we studied DNA samples from patients with various phenotypes of dyslipidemia.

METHODS

Participants (N = 1357) were identified by lipid profiles and screened for the common disease causing LIPA exon 8 skipping splice-site mutation (c.894G>A; p.Ser275_Gln298del; rs116928232).

RESULTS

Six patients were heterozygous for this variant. Complete LIPA sequencing revealed a patient, subsequently confirmed to have LAL D, with a heterozygous frameshift mutation involving deletion of exon 4 (p.Gly77Valfs*17 c.230-106_c.428+541del). A family study revealed a sister with the same genotype and phenotype. Genetic, clinical, and lipoprotein profiles of these sisters plus 6 additional family members are reported. Profiles of 2 other LAL D patients monitored for 2 decades are presented. Cholesterol homeostasis was studied to investigate rates of cholesterol synthesis and absorption in 4 LAL D patients. High-density lipoprotein (HDL) subspecies were also analyzed.

CONCLUSIONS

We used this LIPA sequencing strategy (detection of the relatively common exon 8 variant followed by complete gene sequencing to identify additional mutations) as a means to further elucidate the genetic basis of LAL D among individuals with a suggestive clinical phenotype.

BACKGROUND

Asthma is heterogeneous, and airway dysbiosis is associated with clinical features in patients with mild-to-moderate asthma. Whether similar relationships exist among patients with severe asthma is unknown.

OBJECTIVE

We sought to evaluate relationships between the bronchial microbiome and features of severe asthma.

METHODS

Bronchial brushings from 40 participants in the Bronchoscopic Exploratory Research Study of Biomarkers in Corticosteroid-refractory Asthma (BOBCAT) study were evaluated by using 16S ribosomal RNA-based methods. Relationships to clinical and inflammatory features were analyzed among microbiome-profiled subjects. Secondarily, bacterial compositional profiles were compared between patients with severe asthma and previously studied healthy control subjects (n = 7) and patients with mild-to-moderate asthma (n = 41).

RESULTS

In patients with severe asthma, bronchial bacterial composition was associated with several disease-related features, including body mass index (P < .05, Bray-Curtis distance-based permutational multivariate analysis of variance; PERMANOVA), changes in Asthma Control Questionnaire (ACQ) scores (P < .01), sputum total leukocyte values (P = .06), and bronchial biopsy eosinophil values (per square millimeter, P = .07). Bacterial communities associated with worsening ACQ scores and sputum total leukocyte values (predominantly Proteobacteria) differed markedly from those associated with body mass index (Bacteroidetes/Firmicutes). In contrast, improving/stable ACQ scores and bronchial epithelial gene expression of FK506 binding protein (FKBP5), an indicator of steroid responsiveness, correlated with Actinobacteria. Mostly negative correlations were observed between biopsy eosinophil values and Proteobacteria. No taxa were associated with a TH2-related epithelial gene expression signature, but expression of TH17-related genes was associated with Proteobacteria. Patients with severe asthma compared with healthy control subjects or patients with mild-to-moderate asthma were significantly enriched in Actinobacteria, although the largest differences observed involved a Klebsiella genus member (7.8-fold increase in patients with severe asthma, adjusted P < .001).

CONCLUSIONS

Specific microbiota are associated with and may modulate inflammatory processes in patients with severe asthma and related phenotypes. Airway dysbiosis in patients with severe asthma appears to differ from that observed in those with milder asthma in the setting of inhaled corticosteroid use.

The impact of long-term tamoxifen therapy on ovarian function is not known. Understanding these effects will help reproductive-aged patients who desire future pregnancy make more informed decisions regarding their treatment. This is a retrospective cohort study in patients identified through the UCSF Cancer Registry and SPORE database. We enrolled women with a history of ductal carcinoma in situ (DCIS) or early stage invasive breast cancer who were premenopausal at diagnosis and did not receive chemotherapy. Menstrual histories were obtained through electronic and paper surveys. We compared the age of menopause onset and menstrual pattern changes between women who received tamoxifen (TAM) and those who did not receive tamoxifen (control). Neither group received chemotherapy. 250 subjects were included in this study (125 TAM, 125 control). Mean age of menopause onset was 51.0 for both the groups and was not associated with duration of tamoxifen use or the age at tamoxifen initiation. Menstrual pattern changes, including amenorrhea, were more frequent in the TAM group than control group (any change: 48% TAM vs. 15 % control, p < 0.001; amenorrhea: 22% TAM vs. 3% control, p < 0.001). Older age was associated with an increased risk of developing amenorrhea within 6 months of starting tamoxifen (HR 1.32, p < 0.001). Menstrual pattern changes are common in premenopausal women taking tamoxifen. Tamoxifen use in the absence of chemotherapy is not associated with an earlier age onset of menopause in patients with DCIS or invasive breast cancer and is unlikely to significantly accelerate ovarian aging.