Renin system activation and delayed function of the renal transplant.

Delayed graft function (DGF), defined as persistent renal failure that requires dialysis within the first week after kidney transplantation, occurs commonly after cadaveric renal transplantation (CRT). This has important implications for long-term outcome because the 1-year allograft survival rate is significantly reduced when DGF occurs. The mechanisms contributing to the development of DGF are not well established. However, several lines of evidence indicate that excess renin system activity, in both the cadaver kidney donor and recipient, contributes importantly to the pathogenesis of DGF. If this hypothesis can be verified in clinical studies, then pharmacologic agents that interrupt the renin-angiotensin system (eg, type 1 angiotensin II receptor blockade, angiotensin converting enzyme inhibition, and beta-adrenergic blockade) in the donor and recipient might significantly improve the outcome of cadaveric renal transplants.

Structural basis of neurophysin hormone specificity: Geometry, polarity, and polarizability in aromatic ring interactions.

The structural origins of the specificity of the neurophysin hormone-binding site for an aromatic residue in peptide position 2 were explored by analyzing the binding of a series of peptides in the context of the crystal structure of liganded neurophysin. A new modeling method for describing the van der Waals surface of binding sites assisted in the analysis. Particular attention was paid to the unusually large (5 kcal/mol) difference in binding free energy between Phe and Leu in position 2, a value representing more than three times the maximum expected based on hydrophobicity alone, and additionally remarkable since modeling indicated that the Leu side chain was readily accommodated by the binding pocket. Although evidence was obtained of a weak thermodynamic linkage between the binding interactions of the residue 2 side chain and of the peptide alpha-amino group, two factors are considered central. (1) The bound Leu side chain can establish only one-third of the van der Waals contacts available to a Phe side chain. (2) The bound Phe side chain appears to be additionally stabilized relative to Leu by more favorable dipole and induced dipole interactions with nonaromatic polar and sulfur ligands in the binding pocket, as evidenced by examination of its interactions in the pocket, analysis of the detailed energetics of transfer of Phe and Leu side chains from water to other phases, and comparison with thermodynamic and structural data for the binding of residue 1 side chains in this system. While such polar interactions of aromatic rings have been previously observed, the present results suggest their potential for significant thermodynamic contributions to protein structure and ligand recognition.

Prophylactic use of factor VIII: an economic evaluation.

BACKGROUND: Since the introduction of exogenous factor VIII therapy, several studies have explored the clinical benefits of prophylactic use of factor VIII. Little research, though, has focused on the economic aspects of this regimen. We conducted a cost analysis using data from the Orthopedic Outcomes Study, a prospective, cross-national study of the clinical outcomes associated with different patterns of factor VIII utilization to examine the health care costs incurred and expenditures averted in patients receiving on-demand versus prophylactic use of factor VIII in hemophilia. METHODS AND ANALYSIS: 831 patients with severe hemophilia aged 1 to 31 years, from 19 centers around the world were included in the cost analysis. Patients were categorized into three groups according to the number of weeks during the study years in which they received prophylactic regimens of factor VIII. For each subject, we estimated the costs of hospitalization, surgery, days lost from school or work, and factor VIII utilization. Costs were then stratified by age and by joint score to assess confounding, and a multivariate model developed to determine the relationship between use of factor VIII prophylaxis and total costs, while controlling for potential confounders. RESULTS: Patients who received factor VIII episodically incurred substantially greater disability-related costs (days lost from school or work, days hospitalized due to hemophilia, surgery) than patients who received factor VIII prophylactically for some or all of the study period. For all treatment regimens, most disability-related costs were accounted for by hospitalization for hemophilia-related conditions. The cost of factor VIII itself was substantial in all treatment categories but was highest among patients who received year-round prophylaxis, exceeding the savings resulting from reduced disability and other health care expenditures. CONCLUSIONS: Reductions in non-factor health care costs and disability associated with prophylactic use of factor VIII in hemophilia were substantial and helped somewhat to offset the much higher costs of this regimen. For certain subgroups, frequent episodic treatment may be more expensive than full-time prophylaxis. However, because of the very high cost of year-round prophylactic use of factor VIII, total health care expenditures were highest among patients receiving this therapeutic regimen. However, because prophylaxis clearly offers important clinical benefits, this approach may be warranted on medical rather than economic grounds.

Immunotherapy with anti-CD3 monoclonal antibodies and recombinant interleukin 2: stimulation of molecular programs of cytotoxic killer cells and induction of tumor regression.

Adoptive cellular immunotherapy, infusions of interleukin 2 (IL-2) in conjunction with in vitro-activated killer cells, has brought new hope to patients with cancer. The broad application of this strategy, however, is constrained by the need for repeated leukapheresis and by the labor-intensive process of in vitro activation of cells. Also, current protocols generally use nonphysiological and toxic concentrations of IL-2. Identification of an in vivo stimulant that renders T cells responsive to physiologic concentrations of IL-2 represents a potential improvement over existing approaches. We have determined whether in vivo administration of monoclonal antibodies (mAbs) directed at the T-cell surface protein CD3 induces T-cell responsiveness to IL-2, stimulates cytolytic molecular programs of natural killer cells and cytotoxic T cells, and induces tumor regression. These hypotheses were explored in a murine hepatic MCA-102 fibrosarcoma model. We report that in vivo administration of anti-CD3 mAbs plus IL-2 results in intrahepatic expression of mRNA-encoding perforin, cytotoxic T-cell-specific serine esterase, and tumor necrosis factor alpha. Anti-CD3 mAbs alone or IL-2 alone failed to induce or induced minimal expression of these molecular mediators of cytotoxicity. The anti-CD3 mAbs plus IL-2 regimen also resulted in a significantly smaller number of hepatic metastases and a significantly longer survival time of tumor-bearing mice, compared to treatment with anti-CD3 mAbs alone or IL-2 alone. Our findings suggest that a regimen of anti-CD3 mAbs plus IL-2 is a more effective antitumor regimen compared with anti-CD3 mAbs alone or IL-2 alone and advance an alternative immunotherapy strategy of potential value for the treatment of cancer in humans.

Excellent outcome with a calcium channel blocker-supplemented immunosuppressive regimen in cadaveric renal transplantation. A potential strategy to avoid antibody induction protocols.

Many transplant centers routinely utilize monoclonal antibody or polyclonal antibody based induction protocols in recipients of cadaver renal allografts. Given the potential complications associated with antibody-based immunosuppression regimens (e.g., CMV disease), we tested the hypothesis that a combination of a calcium antagonist and a triple drug protocol (cyclosporine + prednisone + azathioprine) would be an effective substitute for antibody-based induction protocols in ensuring excellent patient and graft survival rates. Our postulate was tested in a prospective study of 52 consecutive recipients of cadaver renal allografts (44 first, 5 second, and 3 third grafts) utilizing nifedipine as the first line calcium antagonist. Nifedipine was selected over verapamil or diltiazem due to its lack of interference with the metabolism of CsA. Some of the significant outcomes of our prospective trial were (A) a cumulative patient survival rate of 98.1% for the 52 recipients at 18 months posttransplantation; (B) a cumulative allograft survival rate of 92.1% for the 52 consecutive cadaver renal allografts at 18 months; (C) a cumulative allograft survival rate of 100% at 18 months for the 24 of 52 renal allografts without delayed graft function following transplantation; and (D) a cumulative allograft survival rate of 86% at 18 months for the 28 of 52 renal allografts with delayed graft function. Of the 4 of 52 who lost their grafts, 2 grafts were removed following discontinuation of immunosuppressive therapy while the remaining 2 had primary nonfunction; and (E) the lack of a requirement for monoclonal or polyclonal antibodies for the treatment of acute rejection episodes in this patient population. These gratifying results compare very favorably with (A) recent reports of the effects of long-term diltiazem therapy and of verapamil used in conjunction with an induction protocol that included Minnesota antilymphocyte globulin in recipients of cadaver renal allografts, and (B) the clinical outcome in many institutions with OKT3/ATG/ALG induction protocols. Whereas the mechanisms involved in the excellent clinical outcome found with the calcium antagonist remain undefined, our results strongly argue for a prospective, randomized and controlled study in which a calcium antagonist-supplemented immunosuppressive regimen is compared with antibody-based induction protocols.

Hypertension in kidney transplant recipients. Effect on long-term renal allograft survival.

To examine the effects of hypertension on renal graft function, we studied the clinical course of 144 kidney transplant recipients who had functioning grafts for three to 13 years. The patients were divided into three groups: normotensive (n = 32), controlled hypertensive (n = 49) and uncontrolled hypertensive group (n = 63). In addition to the difference in their blood pressure status, the three groups had significantly different levels of serum creatinine at entry to the study (mean +/- SE in mg/dL: 1.41 +/- 0.02, 8.89 +/- 0.02 and 2.30 +/- 0.03, respectively, P = .0002). Cumulative graft survival (CGS) at ten years for normotensive patients was 81%, whereas it was 58% for controlled hypertensive patients and 50% for uncontrolled hypertensive patients. The difference of CGS between normotensive and hypertensive patients was significant (P = .01), whereas the difference between the two hypertensive groups, controlled v. uncontrolled, was not. If serum creatinine levels at entry to the study were adjusted and the CGS of hypertensive patients was compared to normotensive patients with comparable levels of serum creatinine, the differences in CGS between the two groups were no longer significant. Regression analyses for potential prognostic factors revealed that serum creatinine levels were of more primary importance as a prognostic variable than blood pressure status. We conclude that hypertension is an important risk factor for renal graft survival, but control of hypertension alone does not appear to improve it. Graft survival appears to be influenced more by the severity of graft dysfunction at entry to the study irrespective of blood pressure control.

In vivo studies of cysteine metabolism. Use of D-cysteinesulfinate, a novel cysteinesulfinate decarboxylase inhibitor, to probe taurine and pyruvate synthesis.

Although several pathways contribute to the catabolism of L-cysteine, the products formed are few--taurine + CO2 and pyruvate + ammonia + sulfate. L-Cysteinesulfinate is a key intermediate that is either decarboxylated to ultimately yield taurine or transaminated to yield pyruvate. There is strong evidence that pyruvate is also formed by several cysteinesulfinate-independent pathways collectively referred to as "cysteine desulfhydrase." The quantitative importance of cysteinesulfinate-independent pathways of taurine synthesis is less clear, but it has been suggested that taurine synthesis from the cysteamine released during phosphopantetheine and CoASH turnover accounts for the high taurine content of tissues with very low levels of cysteinesulfinate decarboxylase activity (e.g. skeletal muscle and heart). In the present studies, the metabolic flux through each of these pathways was quantitated in vivo by monitoring the formation of respiratory 14CO2 in mice administered L-[1-14C]- or L-[3-14C]cyst(e)ine. Mice given 0.05 mmol/kg of L-cystine or 0.5 or 2.5 mmol/kg of L-cysteine catabolize 35, 51, and 72% of the dose, respectively, in 6 h; the relative contribution of taurine synthesis to total catabolism decreases from 63 to 51 to 42% as the L-cyst(e)ine dose is increased. To evaluate the role of L-cysteinesulfinate in taurine synthesis, D-cysteinesulfinate was characterized and used as a metabolism-resistant, potent, and specific inhibitor of cysteinesulfinate decarboxylase. Studies with L-[1-14C]- and L-[3-14C]cysteine in the presence of inhibitor indicate that 85-93% of taurine synthesis occurs from L-cysteinesulfinate: the calculated contribution of the phosphopantetheine pathway is small and may approximate zero. L-Cysteinesulfinate transmamination accounts for 25% of pyruvate synthesis from L-[14C]cystine (0.05 mmol/kg) but only 11% of pyruvate synthesis from L-[14C]cysteine (2.5 mmol/kg). Cysteine desulfhydrase reactions account for most of the pyruvate synthesis.

Generalized finite element solution to one-dimensional flux problems.

A finite element numerical solution to the general one-dimensional flow equation is derived in a form that provides a convenient and general means to simulate a wide variety of one-dimensional flow techniques of interest to biological scientists, e.g., ultracentrifugation, electrophoresis, chromatography, etc. Diverse physical models defined in terms of column geometry, solute interactions, and the dependence of transport parameters on column position, time, or concentrations of one or more solutes, can be accommodated. A particularly useful aspect of the formulation is that a wide variety of boundary conditions can be simply applied to the end result, without rederivation of the solution for each new case. The numerical solution is expressed as matrix equations that are sufficiently general so that incorporation of particular models can be effected by substitution of appropriate quantities into the final result.

Enhanced kidney graft survival with retroplacental source gamma-globulin. Results of a 5-year prospective study at a single center.

We examined the possibility that retroplacental source gamma-globulin (RPGG), with its content of anti-HLA antibodies, would improve cadaver kidney graft survival rates. In a 5-year controlled prospective study of 208 transplants, we found that the addition of RPGG to a standard immunosuppressive drug regimen (azathioprine and prednisone) resulted in significant improvement of the cumulative survival rate (CSR) of first and second grafts. At 2 years, the overall CSR of first grafts increased from a control value of 37% +/- 6 to 52% +/- 6 (P = 0.037). Among second graft recipients, the CSR increased from a value of 19% +/- 8 to 50% +/- 10 (P = 0.014). This improvement in graft survival was seen as early as 3 months after surgery and was sustained through 3 years without added recipient morbidity or mortality. When recipient populations were stratified for various factors, those groupings remonstrative of an intact or active humoral immune response capacity were found to have the highest survival rates in the study; 2-year graft CSRs of 70% +/- 6 and 65% +/- 10 were found in recipients with preformed antibody resulting from blood transfusions (P - 0.003) and cytomegalovirus infectivity (P = 0.0006), respectively. These findings indicate that the improved graft survival seen in this study may have resulted from a recipient's immunological response to challenge with RPGG.

Isolation and characterization of lamellar body material from rat lung homogenates by continuous linear sucrose gradients.

A technique is described for isolating lamellar body material from rat lung. Membranes with relative densities ranging between 1.050 and 1.074 g/ml were isolated by centrifugation of crude lung homogenates upward through continuous linear sucrose gradients at 40,000 rpm (199,000 g) for 3 hr. Their protein and lipid content was characteristic of that of lamellar bodies. They were free of contaminating microsomal and mitochondrial marker enzymes but contained enzyme activities associated with lysosomes and Golgi complex. Longer or repeated centrifugation resulted in a reduced yield and an apparent transformation of some of the material to lower densities. Electron microscopy revealed that most of the images represent disrupted rather than intact lamellar bodies. Other methods for preparation of lamellar bodies entail either sedimentation or pelleting at interfaces between sucrose solutions. Such preparations are often contaminated with endoplasmic reticulum membranes and have apparently lost the more fragile bodies. The present technique reveals the heterogeneous nature of lamellar body material and should be useful in a search for lamellar body precursors and in the investigation of the mechanisms by which surfactant is synthesized or assembled.

General solution to the inverse problem of the differential equation of the ultracentrifuge.

Whenever experimental data can be simulated according to a model of the physical process, values of physical parameters in the model can be determined from experimental data by use of a nonlinear least-squares algorithm. We have used this principle to obtain a general procedure for evaluating molecular parameters of solutes redistributing in the ultracentrifuge that uses time-dependent concentration, concentration-difference, or concentration-gradient data. The method gives the parameter values that minimize the sum of the squared differences between experimental data and simulated data calculated from numerical solutions to the differential equation of the ultracentrifuge.

Electron microscopy of negatively stained and unstained fibrinogen.

Electron microscope images of negatively stained fibrinogen are predominantly asymmetric rods 450 A in length and about 60 A in width. The molecules appear to have considerable flexibility, and mass distribution along the major axis is not uniquely distinguished despite apparent beading in some particles. Scanning transmission electron microscopy of unstained fibrinogen again demonstrates that a majority of molecules are rodlike. The results differ from those obtained by negative staining in that a substantial fraction of images are trinodular with striking resemblance to those obtained by C. E. Hall and H. S. Slayter [J. Biophys. Biochem. Cytol. (1959) 5, 11--16] using the mica replica technique. The above results were obtained on glow-discharged carbon substrate films by a simple low-concentration, long-attachment-time modification of standard deposition methods that is diffusion controlled and depends on concentration and time but is independent of pH, buffer, and other staining conditions. Evidence is presented that standard attachment procedures result in artifactual images. Any models of fibrinogen in solution consequently must encompass properties that permit its visualization as an asymmetetric rod by electron microscopy as first suggested by Hall and Slayter 20 years ago.