Publications

Asthma pathogenesis is focused around conducting airways. The reasons for this focus have been unclear because it has not been possible to track the sites and timing of antigen uptake or subsequent antigen presentation to effector T cells. In this study, we use two-photon microscopy of the lung parenchyma and note accumulation of CD11b(+) dendritic cells (DCs) around the airway after allergen challenge but very limited access of these airway-adjacent DCs to the contents of the airspace. In contrast, we observed prevalent transepithelial uptake of particulate antigens by alveolar DCs. These distinct sites are temporally linked, as early antigen uptake in alveoli gives rise to DC and antigen retention in the airway-adjacent region. Antigen-specific T cells also accumulate in the airway-adjacent region after allergen challenge and are activated by the accumulated DCs. Thus, we propose that later airway hyperreactivity results from selective retention of allergen-presenting DCs and antigen-specific T cells in airway-adjacent interaction zones, not from variation in the abilities of individual DCs to survey the lung.

BACKGROUND

Clinical practice guidelines have been criticized for insufficient attention to the unique needs of patients of advanced age and with multiple comorbid conditions. However, little empiric research is available to inform this topic.

METHODS

We conducted telephone interviews with staff physicians and nurse practitioners in 4 VA health care systems. Respondents were asked to rate the usefulness of national heart failure guidelines for patients of different ages and levels of comorbid burden on a 5-point scale and to comment on the reasons for their ratings.

RESULTS

Of 139 clinicians contacted, 65 (47%) completed the interview. Almost half (49%) were women, and 48 (74%) were general internists or family practitioners. On a 5-point scale assessing the usefulness of clinical practice guidelines for heart failure, the mean (SD) response ranged from 4.4 (0.7) for patients younger than 65 years with few comorbid conditions to 3.5 (1.2) for patients older than 80 years with multiple comorbid conditions (P<0.001). The difference in perceived usefulness varied more by patient age than by degree of comorbidity (P = 0.02). Four major concepts underlay the perceived usefulness of guidelines across different patient types: (1) harm of treatment and complexity of the patient's clinical condition and pharmacologic needs, (2) expected benefits of treatment, (3) patient preferences and abilities, and (4) confidence in the validity of guideline recommendations.

CONCLUSION

Clinicians perceive heart failure guidelines to be substantially less useful in patients of older age and with greater comorbid burden. Concerns about the clinical and pharmacologic complexity of these patients and the expected benefits of drug therapy were commonly invoked as reasons for this skepticism.

Hepatitis B virus (HBV) is a major etiological factor of hepatocellular carcinoma (HCC). However, the precise pathogenetic mechanisms linking HBV infection and HCC remain uncertain. It has been reported that decreased antioxidant enzyme activities are associated with severe liver injury and hepatocarcinogenesis in mouse models. It is unclear if HBV can interfere with the activities of antioxidant enzymes. We established a HBV transgenic mouse line, which spontaneously developed HCC at 2 years of age. We studied the activities of the antioxidant enzymes in the liver of the HBV transgenic mice. Our results showed that the antioxidant enzymes glutathione peroxidase and superoxide dismutase 2 were down-regulated in HBV transgenic mice and correlated with JNK activation. HBV enhanced the Fas-mediated activation of caspase 6, caspase 8 and JNK without enhancing the activation of caspase 3 and hepatocellular apoptosis. As a proper redox balance is important for maintaining cellular homeostasis, these effects of HBV on the host antioxidant system and Fas-signaling may play an important role in HBV-induced hepatocarcinogenesis.

Prostasin is a membrane-anchored protease expressed in airway epithelium, where it stimulates salt and water uptake by cleaving the epithelial Na(+) channel (ENaC). Prostasin is activated by another transmembrane tryptic protease, matriptase. Because ENaC-mediated dehydration contributes to cystic fibrosis (CF), prostasin and matriptase are potential therapeutic targets, but their catalytic competence on airway epithelial surfaces has been unclear. Seeking tools for exploring sites and modulation of activity, we used recombinant prostasin and matriptase to identify substrate t-butyloxycarbonyl-l-Gln-Ala-Arg-4-nitroanilide (QAR-4NA), which allowed direct assay of proteases in living cells. Comparisons of bronchial epithelial cells (CFBE41o-) with and without functioning cystic fibrosis transmembrane conductance regulator (CFTR) revealed similar levels of apical and basolateral aprotinin-inhibitable activity. Although recombinant matriptase was more active than prostasin in hydrolyzing QAR-4NA, cell surface activity resisted matriptase-selective inhibition, suggesting that prostasin dominates. Surface biotinylation revealed similar expression of matriptase and prostasin in epithelial cells expressing wild-type vs. ΔF508-mutated CFTR. However, the ratio of mature to inactive proprostasin suggested surface enrichment of active enzyme. Although small amounts of matriptase and prostasin were shed spontaneously, prostasin anchored to the cell surface by glycosylphosphatidylinositol was the major contributor to observed QAR-4NA-hydrolyzing activity. For example, the apical surface of wild-type CFBE41o- epithelial cells express 22% of total, extractable, aprotinin-inhibitable, QAR-4NA-hydrolyzing activity and 16% of prostasin immunoreactivity. In conclusion, prostasin is present, mature and active on the apical surface of wild-type and CF bronchial epithelial cells, where it can be targeted for inhibition via the airway lumen.

Mutations in the Sonic hedgehog limb enhancer, the zone of polarizing activity regulatory sequence (ZRS, located within the gene LMBR1), commonly called the ZRS), cause limb malformations. In humans, three classes of mutations have been proposed based on the limb phenotype; single base changes throughout the region cause preaxial polydactyly (PPD), single base changes at one specific site cause Werner mesomelic syndrome, and large duplications cause polysyndactyly. This study presents a novel mutation-a small insertion. In a Swedish family with autosomal-dominant PPD, we found a 13 base pair insertion within the ZRS, NG_009240.1:g.106934_106935insTAAGGAAGTGATT (traditional nomenclature: ZRS603ins13). Computational transcription factor-binding site predictions suggest that this insertion creates new binding sites and a mouse enhancer assay shows that this insertion causes ectopic gene expression. This study is the first to discover a small insertion in an enhancer that causes a human limb malformation and suggests a potential mechanism that could explain the ectopic expression caused by this mutation.