The opportunity to do better: lessons old and new from Germany's health system

As the U.S. undertakes national health reform, it would do well to consider both the old and new lessons offered by Germany's century-old universal health system. "Old" principles include an explicit national policy, shared financial responsibility, universality of coverage, and structured financing. New lessons, which are more concerned with operational strategies, include societal affordability, budgets, supply side restrictions, and volume controls.     

Desensitization of guanylyl cyclases in cultured human airway smooth-muscle cells

We previously showed that cultured human airway smooth-muscle cells (HASMC) contain soluble and particulate guanylyl cyclases (GCs). We studied the desensitization of soluble and particulate GCs in HASMC. Homologous desensitization of soluble GC occurred after incubation with S-nitroso-N-acetyl pencillamine (SNAP). SNAP-dependent desensitization was blocked by hemoglobin, a nitric oxide (NO) scavenger, suggesting that it was due to NO release. Cross-desensitization between SNAP and sodium nitroprusside (SNP) and the lack of thiol reduction after SNAP or SNP treatment suggested that thiol depletion was not involved. Assays for soluble GC activity and experiments using protein synthesis inhibitors suggested that SNAP-dependent desensitization was due to reduced soluble GC. Homologous desensitization of particulate GC occurred after pretreatment with atrial natriuretic peptide (ANP) accompanied by reduced particulate GC activity. Recovery required protein synthesis, suggesting that it was due to reduction in particulate GC. Homologous desensitization to either SNAP or ANP was not altered by phosphodiesterase (PDE) inhibitors, suggesting that increased PDE activity was not involved. Cross-desensitization experiments using SNAP and ANP and experiments using zaprinast to elevate cyclic guanosine monophosphate showed no evidence of heterologous desensitization. Our results suggest that pretreatment of HASMC with SNAP or ANP causes homologous, but not heterologous, desensitization of soluble and particulate GCs, respectively.     

Bioelectrical impedance methods in clinical research: a follow-up to the NIH Technology Assessment Conference

In 1994, the National Institutes of Health (NIH) convened a Technology Assessment Conference "to provide physicians with a responsible assessment of bioelectrical impedance analysis (BIA) technology for body composition measurement." In 1997, Serono Symposia USA, Inc., organized an invited panel of scientists and clinicians, with extensive research and clinical experience with BIA, to provide an update. Panel members presented reviews based on their own work and published studies for the intervening years. Updates were provided on the single and multifrequency BIA methods and models; continued clinical research experiences; efforts toward establishing population reference norms; and the feasibility of establishing guidelines for potential diagnostic use of BIA in a clinical setting. This report provides a summary of the panel's findings including a consensus on several technical and clinical issues related to the research use of BIA, and those areas that are still in need of additional study.     

Identification of bacterial pathogens in patients with endophthalmitis by 16S ribosomal DNA typing

PURPOSE: To determine whether sequence analysis of 16S ribosomal DNA (rDNA) can be used to detect bacterial pathogens in patients with postoperative endophthalmitis. METHODS: In 10 eyes of 10 patients, vitreous specimens were collected for culture and rDNA typing. Variable segments of each ribosomal DNA specimen were amplified by polymerase chain reaction (PCR), sequenced, and aligned by BLAST, a computer alignment program, against sequences in GenBank at the National Institutes of Health. RESULTS: Specimens were available from five eyes with bacterial endophthalmitis diagnosed by Gram stain or culture. Amplified 16s rDNA sequences from the eyes of three patients were identical to microbiologic results. Polymerase chain reaction results were negative in two cases in which unusual organisms were detected. All five control specimens from patients with nonbacterial endophthalmitis or uveitis were PCR negative. Approximately 48 to 72 hours are required under ideal conditions for final species identification with this ribosomal typing technique. CONCLUSIONS: 16S rDNA typing shows potential as a relatively rapid technique for identifying bacteria in vitreous samples.     

Molecular characterization of binding of substrates and inhibitors to DT-diaphorase: combined approach involving site-directed mutagenesis, inhibitor-binding analysis, and computer modeling.

The molecular basis of the interaction of DT-diaphorase with a cytotoxic nitrobenzamide CB1954 [5-(aziridin-1-yl)-2, 4-dinitrobenzamide] and five inhibitors was investigated with wild-type DT-diaphorase (human and rat) and five mutants [three rat mutants (rY128D, rG150V, rH194D) and two human mutants (hY155F, hH161Q)]. hY155F and hH161Q were generated to evaluate a hypothesis that Tyr155 and His161 participate in the obligatory two-electron transfer reaction of the enzyme. The catalytic properties of hY155F and hH161Q were compared with a naturally occurring mutant, hP187S. Pro187 to Ser mutation disturbs the structure of the central parallel beta-sheet, resulting in a reduction of the binding affinity of the flavin-adenine dinucleotide prosthetic group. With NADH as the electron donor and menadione as the electron acceptor, the k(cat) values for the wild-type human DT-diaphorase, hY155F, hH161Q, and hP187S were measured as 66 +/- 1, 23 +/- 0, 5 +/- 0 and 8 +/- 2 x 10(3) min(-1), respectively. Because hY155F still has significant catalytic activity, the hydroxyl group on Tyr155 may not be as important as proposed. Interestingly, hY155F was found to be 3. 3 times more active than the human wild-type DT-diaphorase in the reduction of CB1954. Computer modeling based on our results suggests that CB1954 is situated in the active site, with the aziridinyl group pointing toward Tyr155 and the amide group placed near a hydrophobic pocket next to Tyr128. Dicoumarol, Cibacron blue, chrysin, 7,8-dihydroxyflavone, and phenindone are competitive inhibitors of the enzyme with respect to nicotinamide coenzymes. The binding orientations of dicoumarol, flavones, and phenindone in the active site of DT-diaphorase were predicted by results from our inhibitor-binding studies and computer modeling based on published X-ray structures. Our studies generated results that explain why dicoumarol is a potent inhibitor and binds differently from flavones and phenindone in the active site of DT-diaphorase.     

The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)--I. Purification and properties of a nitroreductase enzyme from Escherichia coli--a potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT).

A nitroreductase enzyme has been isolated from Escherichia coli B. This enzyme is an FMN-containing flavoprotein with a molecular mass of 24 kDa and requires either NADH or NADPH as a cofactor. Partial protein sequence analysis showed extensive homology with the "classical nitroreductase" of Salmonella typhimurium and a nitroreductase induced in Enterobacter cloacae. In common with the Salmonella enzyme, the E. coli B enzyme is capable of reducing nitrofurazone. The E. coli nitroreductase is also capable of reducing the anti-tumour agent CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide], a property shared with the mammalian enzyme DT diaphorase [NAD(P)H dehydrogenase (quinone)] as isolated from Walker cells. The reduction of CB1954 by the E. coli enzyme results in the generation of cytotoxic species. Both enzymes also share the properties of being able to reduce quinones and are both inhibited by dicoumarol. The nitroreductase is a more active enzyme against CB1954 (kcat = 360 min-1) than Walker DT diaphorase (kcat = 4 min-1) and also has a lower Km for NADH (6 vs 75 microM).     

Bladder pheochromocytoma encountered on sonography

Pheochromocytomas of the bladder are rare neoplasms, constituting <0.06% of all vesical tumours. Common presenting features of this tumour include episodes of sweating, hypertension, haematuria and postmicturition syncope. We describe a case of bladder pheochromocytoma in a 66‐year‐old man whose only symptom of macroscopic haematuria was initially assessed with ultrasonography. Clinical presentation highlights the need for a high index of suspicion during sonographic evaluation of bladder neoplasms because such tumours might present without symptoms of adrenergic excess.     

Antineoplastic effects of partially HLA-matched irradiated blood mononuclear cells in patients with renal cell carcinoma.

PURPOSE: Vaccines, cytokines, and other biologic-based therapies are being developed as antineoplastic agents. Many of these agents are designed to induce an autologous immune response directed against the malignancy. In contrast, hematopoietic stem-cell transplantation is being developed as a form of allogeneic immunotherapy. This study tests the tolerance and antineoplastic activity of sequential infusions of partially HLA-matched allogeneic blood mononuclear cells (obtained from relatives) when administered outside of the context of a hematopoietic stem-cell transplantation. The cells are irradiated to prevent graft-versus-host disease. PATIENTS AND METHODS: Fifteen patients with relapsed or refractory malignancies for which no standard therapy was available were enrolled onto a clinical trial designed to assess the tolerability and antineoplastic effects of irradiated partially HLA-matched blood mononuclear cells obtained from relatives. RESULTS: There was disease regression in three patients with metastatic renal cell carcinoma during treatment. There was disease progression in six patients with metastatic renal cell carcinoma and two patients with metastatic melanoma during treatment. There was no change in disease state in several other patients. CONCLUSION: Irradiated allogeneic blood mononuclear cells administered outside the context of hematopoietic stem-cell transplantation may induce disease responses in patients with relapsed or refractory malignancies. Transfusion of irradiated allogeneic blood mononuclear cells should be developed further as a novel therapeutic antineoplastic approach.     

Two BRM promoter polymorphisms predict poor survival in patients with hepatocellular carcinoma

Polymorphisms in the promoter of the BRM gene, a critical subunit of the chromatin remodeling SWI/SNF complex, have previously been implicated in risk and prognosis in Caucasian‐predominant lung, head and neck, esophageal, and pancreatic cancers, and in hepatocellular cancers in Asians. We investigated the role of these polymorphisms in hepatocellular carcinoma (HCC) risk and prognosis. HCC cases were recruited in a comprehensive cancer center while the matched controls were recruited from family practice units from the same catchment area. For risk analyses, unconditional logistic regression analyses were performed in HCC patients and matched healthy controls. Overall survival analyses were performed using Cox proportional hazard models, Kaplan‐Meier curves, and log‐rank tests. In 266 HCC cases and 536 controls, no association between either BRM promoter polymorphism (BRM‐741 or BRM‐1321) and risk of HCC was identified (P > 0.10 for all comparisons). There was significant worsening of overall survival as the number of variant alleles increased: BRM‐741 per variant allele adjusted hazards ratio (aHR) 5.77, 95% confidence interval (CI) 2.89‐11.54 and BRM‐1321 per variant allele aHR 4.09, 95%CI 2.22‐7.51. The effects of these two polymorphisms were at least additive, where individuals who were double homozygotes for the variant alleles had a 45‐fold increase in risk of death when compared to those who were double wild‐type for the two polymorphisms. Two BRM promoter polymorphisms were strongly associated with HCC prognosis but were not associated with increased HCC susceptibility. The association was strongest in double homozygotes for the allele variants.