Citations

Directed protein evolution, which employs a combination of random mutagenesis, phage display, and in vitro selection, was used to identify second-site suppressors of the fibronectin (Fn) cell binding domain mutation Asp1495Ala (RGA). The mutations in the Fn 9th (3fn9) and 10th (3fn10) type III repeats obtained after selection on purified integrins αIIbβ3(D119Y) and α5β1 are reported. The 3fn9–10(D1495A) phage with substitution mutations at Asp1418, which is located within the linker region between 3fn9 and 3fn10, enhanced binding to the integrins αIIbβ3 and α5β1, but not αvβ3. The substitution mutations identified at residue Asp1418 were introduced into the native recombinant 3fn9–10 sequence and found to augment binding to αIIbβ3, demonstrating that the observed gain-of-function phenotype was independent of the multivalent character of the phage. These results support the following conclusions. First, regions of Fn in addition to the RGD loop are in close proximity to αIIbβ3 and α5β1 and are capable of participating in the binding to these integrins. Secondly, the conformational relationship between the 3fn9 and 3fn10 modules may be an important factor in the binding of Fn to these two integrins. Thirdly, other altered properties of Fn-integrin interactions, such as integrin specificity, may also be selected. This is the first description of Fn mutations that augment binding to integrins. The ability to select for particular phenotypes in vitro and the subsequent characterization of these mutations should further our understanding of the molecular details involved in the association of integrins and their ligands. Additionally, these higher-affinity 3fn9–10 ligands provide a starting point for further in vitro evolution and engineering of integrin-specific modules.

Abstract We have retrospectively studied 53 patients with idiopathic pulmonary fibrosis and a histologic diagnosis of usual interstitial pneumonia and evaluated the prognostic significance of four individual histologic features (fibroblastic foci [FF], interstitial mononuclear cell infiltrate, established fibrosis, and intra-alveolar macrophages) using a semiquantitative scale of 0–6. An objective count of FF was also undertaken. Using weighted kappa coefficients, interobserver agreement between pathologists was moderate to good (0.56–0.76). Subjective and objective FF scores were strongly associated (RS = 0.88, < 0.00005). Mortality was independently linked to a high FF score, p = 0.006, and a low percent predicted carbon monoxide diffusing capacity (DlCO), p = 0.01. For pulmonary function, on univariate analysis, the strongest correlations were observed between increasing interstitial mononuclear cell infiltrate or FF scores and greater declines in forced vital capacity (FVC) or DlCO at 6 months. Multivariate models revealed that increasing FF scores were independently associated with greater declines in FVC and DlCO at both 6 and 12 months. Increasing interstitial mononuclear cell infiltrate scores were also independently linked to functional decline, but only at 6 months. These data suggest a reproducible method on biopsy for predicting rate of disease progression in patients with idiopathic pulmonary fibrosis.

Abstract Fibrosing alveolitis associated with systemic sclerosis (FASSc) has a better prognosis than idiopathic pulmonary fibrosis. In view of recent reports that idiopathic nonspecific interstitial pneumonia (NSIP) has a better prognosis than idiopathic usual interstitial pneumonia (UIP), we classified histologic appearances of surgical lung biopsies performed in 80 patients with FASSc. NSIP (n = 62, 77.5%), subcategorized as cellular NSIP (n = 15) and fibrotic NSIP (n = 47) was much more prevalent than UIP (n = 6), end-stage lung disease (ESL, n = 6), or other patterns (n = 6). There were 25 deaths (NSIP 16/62, 26%; UIP/ESL 6/12, 50%). Five-year survival differed little between NSIP (91%) and UIP/ESL (82%); mortality was associated with lower initial carbon monoxide diffusing capacity (DlCO) and FVC levels (p = 0.004 and p = 0.007, respectively). Survival and serial FVC and DlCO trends did not differ between cellular and fibrotic NSIP. Increased mortality in NSIP was associated with lower initial DlCO levels (p = 0.04), higher BAL eosinophil levels (p = 0.03), and deterioration in DlCO levels during the next 3 years (p < 0.005). We conclude that NSIP is the histopathologic pattern in most patients with FASSc. However, outcome is linked more strongly to disease severity at presentation and serial DlCO trends than to histopathologic findings.

Background — In patients undergoing percutaneous coronary intervention (PCI) in the modern era, the incidence and prognostic implications of acute renal failure (ARF) are unknown. Methods and Results — With a retrospective analysis of the Mayo Clinic PCI registry, we determined the incidence of, risk factors for, and prognostic implications of ARF (defined as an increase in serum creatinine [Cr] >0.5 mg/dL from baseline) after PCI. Of 7586 patients, 254 (3.3%) experienced ARF. Among patients with baseline Cr <2.0, the risk of ARF was higher among diabetic than nondiabetic patients, whereas among those with a baseline Cr >2.0, all had a significant risk of ARF. In multivariate analysis, ARF was associated with baseline serum Cr, acute myocardial infarction, shock, and volume of contrast medium administered. Twenty-two percent of patients with ARF died during the index hospitalization compared with only 1.4% of patients without ARF ( P <0.0001). After adjustment, ARF remained strongly associated with death. Among hospital survivors with ARF, 1- and 5-year estimated mortality rates were 12.1% and 44.6%, respectively, much greater than the 3.7% and 14.5% mortality rates in patients without ARF ( P <0.0001). Conclusions — The overall incidence of ARF after PCI is low. Diabetic patients with baseline Cr values <2.0 mg/dL are at higher risk than nondiabetic patients, whereas all patients with a serum Cr >2.0 are at high risk for ARF. ARF was highly correlated with death during the index hospitalization and after dismissal.

Background: Antibodies to soluble liver antigen/liver pancreas (SLA/LP) are specific markers of autoimmune hepatitis. Their target antigen has recently been cloned. Aims: To establish standardised immunoassays using the recombinant antigen, and to assess the frequency and significance of seropositivity in patients from different countries. Methods: An enzyme linked immunoassay was developed using purified recombinant antigen and validated by testing sera from 200 healthy blood donors and 1026 patients with various liver and non-liver diseases. The assay was then applied to 454 sera from 419 patients with autoimmune hepatitis from different countries. All sera were also tested by inhibition immunoassay and western blot. Results: Antibodies were reliably detected by the recombinant immunoassay and occurred exclusively in patients with autoimmune liver disease. Twenty three of 149 patients from the USA (15%), 23/132 from Brazil (17%), 21/108 from Germany (19%), and 2/30 from Japan (7%) were seropositive. Clinical features at presentation were similar between seropositive and seronegative patients. However, relapse after corticosteroid withdrawal or during maintenance therapy occurred more commonly in seropositive patients. Conclusions: Antibodies to SLA/LP can be reliably detected by these standardised immunoassays based on recombinant antigen. Antibodies to SLA/LP occur with similar frequencies in different geographical regions, races, and age groups, and are of exquisite diagnostic specificity. Whether SLA/LP positive patients represent a clinically distinct subgroup remains to be determined; relapse during treatment reduction appeared to be more common in the SLA/LP group.

Background and aims: This was a prospective blinded study to compare computed tomography (CT) colonography, performed with multidetector arrays CT scan (MDCT), with conventional colonoscopy for the detection of colorectal neoplasia. Methods: Fifty patients were examined by MDCT after standard bowel preparation and rectal air insufflation in the supine and prone positions. Data sets were examined by one radiologist and one gastroenterologist blinded to the patient's history and colonoscopy results. Patients subsequently underwent colonoscopy on the same day, which served as the gold standard. Results: Nine of 11 lesions >10 mm (82%), 5/15 lesions of 6–9 mm (33%), and 1/42 polyps <5 mm (3%) were detected by MDCT colonography. One false positive result for a structure larger than 10 mm was described. Nineteen of 21 patients who had no lesions during conventional colonoscopy were considered free of lesions by MDCT colonography, yielding a per patient specificity of 90%. Conclusion: MDCT colonography provides good data quality and has good sensitivity and specificity for the detection of colonic lesions of 10 mm or more.

ABSTRACT Intracellular calcium signaling plays a central role in cell proliferation. In leukemic cells, the calcium release‐activated calcium channels provide a major pathway for calcium entry (I CRAC ) perpetuating progression through the cell cycle. Although I CRAC is under mitochondrial regulation, targeting mitochondrial function has not been exploited to control malignant cell growth. The benzothiadiazine diazoxide, which depolarized respiration‐dependent mitochondrial membrane potential, reduced the rate of proliferation and arrested human acute leukemic T cells in the G0/G1 phase. Diazoxide did not alter cellular energetics, but rather inhibited the mitochondria‐controlled I CRAC and reduced calcium influx into tumor cells. The antiproliferative action of diazoxide was mimicked by removal of extracellular calcium or by the tyrphostin A9, an I CRAC inhibitor. Deletion of the mitochondrial genome, which encodes essential respiratory chain enzyme subunits, attenuated the inhibitory effect of diazoxide on I CRAC ‐mediated calcium influx and cell proliferation. Thus, manipulation of mitochondrial function and associated calcium signaling provides a basis for a novel anticancer strategy.—Holmuhamedov, E., Lewis, L., Bienengraeber, M., Holmuhamedova, M., Jahangir, A., Terzic, A. Suppression of human tumor cell proliferation through mitochondrial targeting. FASEB J. 16, 1010–1016 (2002)