Utility of oropharyngeal real-time PCR for S. pneumoniae and H. influenzae for diagnosis of pneumonia in adults.

A lack of sensitive tests and difficulties obtaining representative samples contribute to the challenge in identifying etiology in pneumonia. Upper respiratory tract swabs can be easily collected and analyzed with real-time PCR (rtPCR). Common pathogens such as S. pneumoniae and H. influenzae can both colonize and infect the respiratory tract, complicating the interpretation of positive results. Oropharyngeal swabs were collected (n = 239) prospectively from adults admitted to hospital with pneumonia. Analysis with rtPCR targeting S. pneumoniae and H. influenzae was performed and results compared with sputum cultures, blood cultures, and urine antigen testing for S. pneumoniae. Different Ct cutoff values were applied to positive tests to discern colonization from infection. Comparing rtPCR with conventional testing for S. pneumoniae in patients with all tests available (n = 57) resulted in: sensitivity 87 %, specificity 79 %, PPV 59 % and NPV 94 %, and for H. influenzae (n = 67): sensitivity 75 %, specificity 80 %, PPV 45 % and NPV 94 %. When patients with prior antimicrobial exposure were excluded sensitivity improved: 92 % for S. pneumoniae and 80 % for H. influenzae. Receiver operating characteristic curve analysis demonstrated for S. pneumoniae: AUC = 0.65 (95 % CI 0.51-0.80) and for H. influenzae: AUC = 0.86 (95 % CI 0.72-1.00). Analysis of oropharyngeal swabs using rtPCR proved both reasonably sensitive and specific for diagnosing pneumonia caused by S. pneumoniae and H. influenzae. This method may be a useful diagnostic adjunct to other methods and of special value in patients unable to provide representative lower airway samples.

Cystic fibrosis transmembrane conductance regulator Cl- channels with R domain deletions and translocations show phosphorylation-dependent and -independent activity.

Phosphorylation of the R domain regulates cystic fibrosis transmembrane conductance regulator Cl- channel activity. Earlier studies suggested that the R domain controls activity via more than one mechanism; a phosphorylated R domain may stimulate activity, and an unphosphorylated R domain may prevent constitutive activity, i.e. opening with ATP alone. However, the mechanisms responsible for these two regulatory properties are not understood. In this study we asked whether the two effects are dependent on its position in the protein and whether smaller regions from the R domain mediate the effects. We found that several portions of the R domain conferred phosphorylation-stimulated activity. This was true whether the R domain sequences were present in their normal location or were translocated to the C terminus. We also found that some parts of the R domain could be deleted without inducing constitutive activity. However, when residues 760-783 were deleted, channels opened without phosphorylation. Translocation of the R domain to the C terminus did not prevent constitutive activity. These results suggest that different parts of the phosphorylated R domain can stimulate activity and that their location within the protein is not critical. In contrast, prevention of constitutive activity required a short specific sequence that could not be moved to the C terminus. These results are consistent with a recent model of an R domain composed primarily of random coil in which more than one phosphorylation site is capable of stimulating channel activity, and net activity reflects interactions between multiple sites in the R domain and the rest of the channel.

Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia.

The regulatory domain of cystic fibrosis transmembrane conductance regulator (CFTR) regulates channel activity when several serines are phosphorylated by cAMP-dependent protein kinase. To further define the functional role of individual phosphoserines, we studied CFTR containing previously studied and new serine to alanine mutations. We expressed these constructs in Fischer rat thyroid epithelia and measured transepithelial Cl(-) current. Mutation of four in vivo phosphorylation sites, Ser(660), Ser(737), Ser(795), and Ser(813) (S-Quad-A), substantially decreased cAMP-stimulated current, suggesting that these four sites account for most of the phosphorylation-dependent response. Mutation of either Ser(660) or Ser(813) alone significantly decreased current, indicating that these residues play a key role in phosphorylation-dependent stimulation. However, neither Ser(660) nor Ser(813) alone increased current to wild-type levels; both residues were required. Changing Ser(737) to alanine increased current above wild-type levels, suggesting that phosphorylation of Ser(737) may inhibit current in wild-type CFTR. These data help define the functional role of regulatory domain phosphoserines and suggest interactions between individual phosphoserines.

A functional R domain from cystic fibrosis transmembrane conductance regulator is predominantly unstructured in solution.

Phosphorylation of the regulatory (R) domain initiates cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel activity. To discover how the function of this domain is determined by its structure, we produced an R domain protein (R8) that spanned residues 708-831 of CFTR. Phosphorylated, but not unphosphorylated, R8 stimulated activity of CFTR channels lacking this domain, indicating that R8 is functional. Unexpectedly, this functional R8 was predominantly random coil, as revealed by CD and limited proteolysis. The CD spectra of both phosphorylated and nonphosphorylated R8 were similar in aqueous buffer. The folding agent trimethylamine N-oxide induced only a small increase in the helical content of nonphosphorylated R8 and even less change in the helical content of phosphorylated R8. These data, indicating that the R domain is predominantly random coil, may explain the seemingly complex way in which phosphorylation regulates CFTR channel activity.

Double jeopardy: lung cancer after cardiac transplantation.

Two heart transplant patients were referred on the same day for evaluation of new chest radiograph abnormalities. Each proved to have advanced stage bronchogenic carcinoma. Review of the recent medical literature reveals that the combination of profound immunosuppression and a heavy smoking history puts cardiac transplant recipients at increased risk for the development of aggressive lung cancers.

Giant cell arteritis in Iceland. An epidemiologic and histopathologic analysis.

OBJECTIVE: To investigate the incidence and clinical and histopathologic features of giant cell (temporal) arteritis (GCA) in the Caucasian population of Iceland. METHODS: All patients diagnosed between 1984 and 1990 were included. Case ascertainment for the study was done in 2 ways: 1) a computerized search from all hospitals and primary care clinics for the diagnosis of GCA, and 2) a review of all temporal artery biopsies performed during the 7-year period. RESULTS: One hundred thirty-three patients with GCA were identified. All fulfilled the 1990 American College of Rheumatology criteria for the classification of GCA. The incidence rate for the population 50 years and older was 27/100,000 (36/100,000 and 18/100,000 for women and men, respectively). Clinical findings included the following: mean age at diagnosis 72.5 years for women and 70.3 years for men, new headache 63.2%, abnormal temporal artery on palpation 43.6%, mean erythrocyte sedimentation rate 88 mm/hour, symptoms of polymyalgia rheumatica 48.1%, and visual disturbances 14.3%. A total of 744 patients underwent temporal artery biopsy during the 7-year period; 16.8% had a positive biopsy result. All 133 patients with the diagnosis of GCA underwent a temporal artery biopsy; 94% had a positive result. Histopathologic findings from the positive biopsies included a fragmented internal elastic lamina in 99.2%, giant cells in 65.6%, and fibrinoid necrosis in 12%. CONCLUSION: Compared with previous epidemiologic surveys, this study shows a high incidence of biopsy-proven GCA in Iceland.